Cancer methods

ABSTRACT

A method of treating a patient having a cancer in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is upregulated, the method comprising administering to the patient a compound comprising or consisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereof which comprises at least one Ig domain of OPCML, or a variant thereof having at least 90% sequence identity with the OPCML polypeptide or the fragment thereof, or a nucleic acid molecule which encodes the OPCML polypeptide or fragment or variant thereof.

This application is a national stage application under 35 U.S.C. 371 ofPCT Application No. PCT/GB2011/050754, filed Apr. 15, 2011, which claimsthe priority benefit of U.S. Provisional Patent Application Ser. No.61/325,013, filed Apr. 16, 2010, and Great Britain Patent ApplicationSerial No. 1105584.5, filed Apr. 1, 2011 which are hereby incorporatedby reference in their entirety.

The present invention relates to the field of medicine, morespecifically to the field of cancer. In particular, the presentinvention relates to the role of OPCML in cancer prognosis, theragnosis,treatment and research.

Cancer is a leading cause of mortality worldwide, accounting for 7.9million deaths (around 13% of all deaths) in 2007, with lung, stomach,liver, colon and breast cancers causing the most deaths each year (WorldHealth Organization, WHO). According to the to WHO, deaths from cancerworldwide are projected to continue rising, with an estimated 12 milliondeaths in 2030. There is thus a very great need for additional andimproved methods of treating cancers.

The etiology of cancer is multifactorial, with genetic, environmental,medical, and lifestyle factors interacting to produce a givenmalignancy. Knowledge of cancer genetics is rapidly improving ourunderstanding of cancer biology, helping to identify at-riskindividuals, furthering the ability to characterize malignancies,establishing treatment tailored to the molecular fingerprint of thedisease, and leading to the development of new therapeutic modalities.Thus there is a need for greater knowledge and understanding of cancergenetics in order to improve all aspects of cancer management, includingprevention, screening, and treatment.

OPCML (also known as OBCAM) is a member of the IgLON family ofcell-surface GPI-anchored proteins and has been previously validated asan epigenetic biomarker for ovarian cancer. In WO 03/002765, and inSellar et al (2003, Nature Genetics 34(3): 337-343), we showed that theOPCML gene shows frequent loss of heterozygosity in human tumors, thatOPCML gene inactivation is via extensive methylation of a CpG island inthe promoter, and that OPCML behaves as a tumor suppressor gene in invivo models.

Subsequently, OPCML was found to be epigenetically inactivated anddownregulated in a wide a variety of cancers. For example, Reed et alfound that OPCML is down-regulated in gliomas and other brain tumors(Reed et al (2007) “Expression of cellular adhesion molecule ‘OPCML’ isdown-regulated in gliomas and other brain tumors” Neuropathology andApplied Neurobiology 33(1): 77-85), Cui et al found that the expressionof OPCML is frequently silenced or down-regulated in multiple tumors dueto its high level of promoter methylation (Cui et al (2008) “OPCML Is aBroad Tumor Suppressor for Multiple Carcinomas and Lymphomas withFrequently Epigenetic Inactivation”, PLoS ONE 3(8): e2990), and Li et alfound OPCML methylation in hepatocellular carcinoma (Li et al (2010)“CpG Island Methylator Phenotype Associated with Tumor Recurrence inTumor-Node-Metastasis Stage I Hepatocellular Carcinoma” Ann. Surg.Oncol. [Epub ahead of print]).

We have now found that OPCML is a genetic marker of cancer that can beused, alone or in combination with other genetic markers, for theprognosis of cancer in human patients and to assess progression freesurvival (PFS). Our studies on the role and effect of OPCML on receptortyrosine kinases (RTKs) have shown that OPCML can be used as a geneticmarker to select specific treatments for cancer patients. We have alsoshown that OPCML can be used as treatment for specific subsets ofpatients defined by their RTK profile.

Specifically, we have shown that OPCML mediates a growth suppressiveeffect by negatively regulating specific receptor tyrosine kinases,especially HER2, HER4, FGFR1, EPHA2 and FGFR3, and we highlightprognostic, predictive and therapeutic approaches based on OPCML's rolein cancer. In Example 1 we demonstrate the effect of OPCML on receptortyrosine kinase signaling in OPCML expressing cell lines and show thatOPCML is a prognostic factor for ovarian cancer; in Example 2 we provideevidence that exogenous OPCML inhibits proliferation of cancer celllines in vitro; in Example 3 we confirm that OPCML is also a prognosticfactor for breast, lung and glioma cancers; in Example 4 we provideevidence of the effect of OPCML on inhibiting cancer cell growth invivo; and in Example 5 we provide further supporting evidence of theeffects of OPCML.

Accordingly, a first aspect of the invention provides a method oftreating a patient having a cancer in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 is upregulated and/or in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 mediated-signaling is upregulated, the method comprisingadministering to the patient a compound comprising or consisting of anOPCML polypeptide (SEQ ID NO: 1), or a fragment thereof which comprisesat least one Ig domain of OPCML, or a variant thereof having at least90% sequence identity with the OPCML polypeptide or the fragmentthereof, or a nucleic acid molecule which encodes the OPCML polypeptideor fragment or variant thereof.

This aspect of the invention includes the use of a compound comprisingor consisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragmentthereof which comprises at least one Ig domain of OPCML, or a variantthereof having at least 90% sequence identity with the OPCML polypeptideor the fragment thereof, or a nucleic acid molecule which encodes theOPCML polypeptide or fragment or variant thereof, in the preparation ofa medicament for treating a patient having a cancer in which HER2, HER4,FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which HER2, HER4,FGFR1, EPHA2 and/or FGFR3 mediated-signaling is upregulated.

This aspect of the invention also includes a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises at least one Ig domain of OPCML, or a variant thereofhaving at least 90% sequence identity with the OPCML polypeptide or thefragment thereof, or a nucleic acid molecule which encodes the OPCMLpolypeptide or fragment or variant thereof for use in treating a patienthaving a cancer in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3 isupregulated and/or in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3mediated-signaling is upregulated.

The cDNA and amino acid sequences of human HER2 (aka ERBB2; Entrez GeneID: 2064) are publicly available from the GenBank database underAccession Nos. NM_004448 and NP_004439.

The cDNA and amino acid sequences of human HER4 (aka ERBB4; Entrez GeneID: 2066) are publicly available from the GenBank database underAccession Nos. NM_005235 and NP_005226.

The cDNA and amino acid sequences of human FGFR1 (Entrez Gene ID: 2260)are publicly available from the GenBank database under Accession Nos.NM_023110 and NP_075598.

The cDNA and amino acid sequences of human FGFR3 (Entrez Gene ID: 2261)are publicly available from the GenBank database under Accession Nos.NM_000142 and NP_000133.

The cDNA and amino acid sequences of human EPHA2 (Entrez Gene ID: 1969)are publicly available from the GenBank database under Accession Nos.NM_004431 and NP_004422 (update version 20 Mar. 2011).

The upregulation of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 may bedetermined by assessing HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mRNAlevels, polypeptide levels or gene copy number using methods that arewell known in the art.

In an embodiment, by a cancer in which HER2, HER4, FGFR1, EPHA2 and/orFGFR3 is upregulated we mean that the HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mRNA or polypeptide levels in the cancer are at least 2× greaterthan control levels. Preferably, the HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mRNA or polypeptide levels are at least 3×, or at least 4×, or atleast 5×, or at least 10×, or at least 20×, or at least 30×, or at least40×, or at least 50×, or at least 100× greater than control levels. TheHER2, HER4, FGFR1, EPHA2 and/or FGFR3 mRNA or polypeptide levels in thecancer may be at least 500×, or at least 1,000×, or at least 10,000×greater than control levels.

In an embodiment, by a cancer in which HER2, HER4, FGFR1, EPHA2 and/orFGFR3 is upregulated, we mean that the HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mRNA or polypeptide levels in the cancer are above-median, andpreferably are upper quartile, compared to control levels.

In an embodiment, the control levels for HER2, HER4, FGFR1, EPHA2 and/orFGFR3 in the cancer of a specific tissue or organ may be the average(mean, or preferably median) level of HER2, HER4, FGFR1, EPHA2 and/orFGFR3 found in the same tissue or organ in a population of individualswho do not have cancer in that tissue or organ, and typically do nothave cancer at all. Typically, the population of individuals is matchedfor gender, age, and ethnic origin. Preferably, the population ofcontrol individuals comprises at least 5, 10, 50, 100, 200, 300, 400 or500 individuals and more preferably at least 1000, 5000 or 10000individuals.

In an alternative embodiment, the control levels for HER2, HER4, FGFR1and/or FGFR3 in the cancer of a specific tissue or organ may be theaverage (mean, or preferably median) level of HER2, HER4, FGFR1 and/orFGFR3 found in a population of patients with the same cancer. Typically,the population of individuals is matched for gender, age, and ethnicorigin. Preferably, the population of control individuals comprises atleast 5, 10, 50, 100, 200, 300, 400 or 500 individuals and morepreferably at least 1000, 5000 or 10000 individuals.

In an specific embodiment, the level of HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mRNA or polypeptide that is indicative of upregulation is a levelof HER2, HER4, FGFR1, EPHA2 and/or FGFR3 in the tissue or organ with thecancer that is equal to or greater than the mean+2 standard deviations(SD) of the level of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mRNA orpolypeptide found in a population of control individuals, usually thecontrol individuals without cancer.

The HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mRNA levels may be measured bymethods such as quantitative RT-PCR, as is well known in the art.

In another embodiment, the HER2, HER4, FGFR1, EPHA2 and/or FGFR3polypeptide levels may be measured by immunohistochemistry (IHC). UsingHercepTest™, a semi-quantitative immunohistochemical assay fordetermination of HER2 protein (c-erbB-2 oncoprotein) overexpression incancer tissues, HER2 is routinely graded as 0, 1+, 2+, or 3+. If theresults are 3+ the cancer is considered to be HER2-positive (HER2+). Forthe avoidance of doubt, in the context of this invention a HER2+ canceris one in which HER2 is upregulated.

A similar IHC grading scale may be used for HER4, FGFR1, EPHA2 and FGFR3wherein a 3+ grading indicates that the cancer is HER4+, FGFR1+, EPHA2+or FGFR3+, and that the cancer has upregulated HER4, FGFR1, EPHA2 orFGFR3, respectively.

Alternatively, the HER2, HER4, FGFR1, EPHA2 and/or FGFR3 gene copynumber may be measured by Fluorescence In Situ Hybridization (FISH).This test uses fluorescent probes to look at the number of gene copiesin a cancer cell. If there are more than two copies of the HER2 or HER4gene, then the cancer is HER2+ or HER4+ positive, and is considered tohave upregulated HER2 or HER4, respectively.

For FGFR1 and FGFR3, if >50% of the neoplastic cells have >5 copies ofthe gene or large gene clusters, the cancer is considered to be FGFR1+or FGFR3+, and may be considered to have upregulated FGFR1+ or FGFR3+,respectively (Elsheikh et al (2007) “FGFR1 amplification in breastcarcinomas: a chromogenic in situ hybridisation analysis” Breast CancerResearch 9: R23).

EPHA2 upregulation can readily be determined using methods well known inthe art, such as those described, for example, by Kataoka et al (2004)“Correlation of EPHA2 overexpression with high microvessel count inhuman primary colorectal cancer.” Cancer Sci. 95: 136-141; Miyazaki etal (2003) “EphA2 overexpression correlates with poor prognosis inesophageal squamous cell carcinoma.” Int J. Cancer. 103: 657-663; andNakamura et al (2005) “EPHA2/EFNA1 expression in human gastric cancer.”Cancer Sci. 96: 42-47.

Thus, by a cancer in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3 isupregulated, we also mean that the cancer is considered to ‘positive’for HER2, HER4, FGFR1, EPHA2 and/or FGFR3.

In an embodiment, the upregulation of HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mediated-signaling may be determined by assessing HER2, HER4,FGFR1, EPHA2 and/or FGFR3 ligand levels in the patient. EPHRINA1 (EFNA1)is the ligand for EPHA2. Increased ligand levels, e.g. 2×, 5×, 10×, 50×or 100× above control levels, or above-median or upper-quartile levels,compared to control levels, may be indicative that the RTKmediated-signaling is upregulated. Ligand levels can be determined, forexample, using methods that are well known in the art such as FISH tomeasure gene copy number, qRT-PCR, to measure mRNA levels andsemi-quantitative protein evaluation by IHC or Western blotting.

Cancers in which HER2, HER4, FGFR1, EPHA2 and/or FGFR3mediated-signaling is upregulated may be determined by various methodsin the art. For example, the expression of HER2, HER4, FGFR1, EPHA2and/or FGFR3 may be assayed in any biological sample that is directly orindirectly derived from the patient such as a cancer biopsy or extract.In an embodiment, antibody-based techniques may be preferred. The normalrange of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 expression can then bedefined using values from either healthy individuals, or a population ofindividuals with the same cancer, which can be compared to thoseobtained from a test patient. Alternatively, the level of HER2, HER4,FGFR1, EPHA2 and/or FGFR3 mediated-signaling can be determined byassessing downstream targets. For example, a particular protein whoseexpression is known to be under the control of the HER2, HER4, FGFR1,EPHA2 and/or FGFR3 signaling pathway(s) may be quantified either at thenucleic acid or protein level, typically by qRT-PCR, IHC or Westernblotting.

In some cancers, a mutation in the ligand-binding domain of HER2, HER4,FGFR1, EPHA2 and/or FGFR3 may cause constitutive activation of thereceptor in the absence of ligand binding, and thus the upregulation ofHER2, HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling (Arteaga (2002)“Epidermal growth factor receptor dependence in human tumors: more thanjust expression?” Oncologist 7: Suppl 4: 31-39). Mutations that causeconstitutive activation of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 can bedetermined, for example, using methods that are well known in the art,such as DNA sequencing of constitutively active receptors.

In an embodiment, by a cancer in which HER2, HER4, FGFR1, EPHA2 and/orFGFR3 mediated-signaling is upregulated we also include a cancer that isaddicted to a HER2, HER4, FGFR1, EPHA2 or FGFR3 signaling pathway. As isknown in the art, when a cancer is ‘addicted’ to a specific RTK pathway,such as a HER2, HER4, FGFR1, EPHA2 or FGFR3 signaling pathway, thecancer becomes largely reliant on the single activated oncogene forcellular signaling (Weinstein & Joe (2008) “Oncogene addiction” CancerRes. 68: 3077-3080; Weinstein & Joe (2006) “Mechanisms of disease:oncogene addiction—a rationale for molecular targeting in cancertherapy” Nat Clin Pract Oncol. 3: 448-457; Sharma et al (2006)“Oncogenic shock: explaining oncogene addiction through differentialsignal attenuation” Clin Cancer Res. 12: 4392s-4395s).

The addiction of a cancer to the HER2, HER4, FGFR1, EPHA2 or FGFR3pathway may be determined by methods well known in the art, such as copynumber estimation via SNP chip or by comparative genomic hybridization(CGH), by IHC/Western blotting, or by qRT-PCR.

In an embodiment, the method may further comprise the prior step ofidentifying a patient having a cancer in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 is upregulated and/or in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 mediated-signaling is upregulated. Typically, this involvesidentifying a patient having a cancer in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 is upregulated and/or in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 mediated-signaling is upregulated from the results ofpreviously-conducted testing.

In an alternative embodiment, the method may further comprise the priorstep of determining whether the patient has a cancer in which HER2,HER4, FGFR1, EPHA2 and/or FGFR3 is upregulated, and/or in which HER2,HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is upregulated.Typically, this involves testing a sample from the patient to determinewhether the patient has a cancer in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 is upregulated, and/or in which HER2, HER4, FGFR1, EPHA2and/or FGFR3 mediated-signaling is upregulated.

OPCML (aka OBCAM) was described in 1995 by Shark & Lee (Shark & Lee(1995) “Cloning, sequencing and localization to chromosome 11 of a cDNAencoding a human opioid-binding cell adhesion molecule (OBCAM)” Gene155: 213-217). The cDNA and amino acid sequences of human OPCML arepublicly available from GenBank under Accession Nos. NM_002545 andNP_002536. The amino acid sequence from NP_002536 is also shown in FIG.14 (SEQ ID NO: 1). Further sequences for OPCML in rat and cow areavailable from GenBank under the following Accession Nos: M88711 (Rattusnorvegicus) and X12672 (Bos taurus).

The Ig1 domain of OPCML spans residues 39-126 of human OPCML, the Ig2domain of OPCML spans residues 136-219 of human OPCML, and the Ig3domain of OPCML spans residues 223-310 of human OPCML. The amino acidsequence of the Ig1 domain (SEQ ID No: 4), Ig2 domain (SEQ ID No: 2) andIg3 domain (SEQ ID No: 3) of human OPCML are listed in FIG. 15.

In a preferred embodiment, the fragment of OPCML may be one which islacking the signal sequence, which is found at residues 1-27 of humanOPCML.

Additionally or alternatively preferably, the fragment of OPCML may beone which is lacking the C-terminal GPI-anchor site, which is at N322,and residues 323-345 which are cleaved in the attachment process. Wehave shown that recombinant OPCML lacking this site, i.e. lackingresidues 322-345, has activity in in vitro and in vivo studies. This isparticularly surprising as it would not expected that the recombinantOPCML protein would have activity if it lacked its membrane attachmentsite. Thus, it is appreciated that the invention includes apharmaceutical composition comprising a fragment of OPCML, preferablyhuman OPCML, that lacks the GPI-anchor site; its use as a medicament;the use of such a fragment of OPCML in the preparation of a medicamentfor treating cancer; a method of treating cancer by administering such afragment of OPCML to a patient; and such a fragment of OPCML for use intreating cancer.

Accordingly, in an embodiment, the fragment of OPCML for use in theabove methods may be one that comprises or consists of residues 28-321of human OPCML. It is appreciated that the invention includes apharmaceutical composition comprising such a fragment of human OPCML;its use as a medicament; the use of such a fragment of OPCML in thepreparation of a medicament for treating cancer; a method of treatingcancer by administering such a fragment of OPCML to a patient; and sucha fragment of OPCML for use in treating cancer.

In a further embodiment, the fragment of OPCML for use in the abovemethods may be one that comprises or consists of residues 39-310 ofhuman OPCML. It is also appreciated that the invention includes apharmaceutical composition comprising such a fragment of human OPCML;its use as a medicament; the use of such a fragment of OPCML in thepreparation of a medicament for treating cancer; a method of treatingcancer by administering such a fragment of OPCML to a patient; and sucha fragment of OPCML for use in treating cancer.

In another embodiment, the fragment of OPCML for use in the abovemethods may be one that comprises or consists of residues 39-219 ofhuman OPCML. It is also appreciated that the invention includes apharmaceutical composition comprising such a fragment of human OPCML;its use as a medicament; the use of such a fragment of OPCML in thepreparation of a medicament for treating cancer; a method of treatingcancer by administering such a fragment of OPCML to a patient; and sucha fragment of OPCML for use in treating cancer.

In yet another embodiment, the fragment of OPCML for use in the abovemethods may be one that comprises or consists of residues 39-126 andresidues 223-310 of human OPCML. It is also appreciated that theinvention includes a pharmaceutical composition comprising such afragment of human OPCML; its use as a medicament; the use of such afragment of OPCML in the preparation of a medicament for treatingcancer; a method of treating cancer by administering such a fragment ofOPCML to a patient; and such a fragment of OPCML for use in treatingcancer.

In still yet another embodiment, the fragment of OPCML for use in theabove methods may be one that comprises or consists of residues 136-310of human OPCML. It is also appreciated that the invention includes apharmaceutical composition comprising such a fragment of human OPCML;its use as a medicament; the use of such a fragment of OPCML in thepreparation of a medicament for treating cancer; a method of treatingcancer by administering such a fragment of OPCML to a patient; and sucha fragment of OPCML for use in treating cancer.

It is appreciated that a fragment of OPCML that comprises a specifiedregion of OPCML may contain additional residues of OPCML outside thespecified region, but does not include the full length protein. Morepreferably, if the fragment of OPCML comprises a specified region ofOPCML and contains additional residues of OPCML outside the specifiedregion, it does not include the GPI-anchor site, the signal sequence oreither of the GPI-anchor site and the signal sequence.

It is also appreciated that a fragment of OPCML that comprises orconsists of a specified region of OPCML may be in the form of a fusionmolecule, as discussed below.

In an embodiment, the fragment of OPCML is at least 85 amino acids inlength, such as at least 90, at least 100, at least 125, at least 150,at least 175, at least 200, at least 225, at least 250, at least 275, atleast 300 or at least 325 amino acids in length.

In an embodiment, the variant of the OPCML polypeptide or the fragmentthereof has at least 91% sequence identity, or at least 92% sequenceidentity, or at least 93% sequence identity, or at least 94% sequenceidentity, or at least 95% sequence identity, or at least 96% sequenceidentity, or at least 97% sequence identity, or at least 98% sequenceidentity, or at least 99% sequence identity, with the OPCML polypeptideor the fragment thereof. Such fragments and variants may be made, forexample, using the methods of recombinant DNA technology, proteinengineering and site-directed mutagenesis, which are well known in theart, and discussed in more detail below.

The percent sequence identity between two polypeptides may be determinedusing suitable computer programs, for example the GAP program of theUniversity of Wisconsin Genetic Computing Group and it will beappreciated that percent identity is calculated in relation topolypeptides whose sequence has been aligned optimally. The alignmentmay alternatively be carried out using the Clustal W program (Thompsonet al., (1994) Nucleic Acids Res 22, 4673-80). The parameters used maybe as follows: Fast pairwise alignment parameters: K-tuple(word) size;1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoringmethod: x percent. Multiple alignment parameters: gap open penalty; 10,gap extension penalty; 0.05. Scoring matrix: BLOSUM.

It is appreciated that OPCML mutations, albeit infrequent, have beendetected in ovarian cancer, some of which somatic mutations have beeninactivating and lead to a complete loss of function. It is thuspreferred that the variant of OPCML or the fragment thereof does notpossess any of the mutations that are known to be inactivating of OPCMLfunction. In particular, it is preferred that the variant of OPCML orthe fragment thereof does not have a P95R somatic mutation whichdemonstrates loss of components of domain 1 function, such asintercellular adhesion and cell-matrix binding inhibition on collagensubstrates.

It is preferred that the fragment of OPCML, or the variant of the OPCMLor fragment thereof possesses at least 50% of the activity of fulllength human OPCML in inhibiting the proliferation of cancer cells invitro. It is more preferred if the fragment of OPCML, or the variant ofthe OPCML or fragment thereof possesses at least 50%, or at least 60%,or at least 70%, or at least 80%, or at least 90%, or at least 100% ormore of the activity of full length human OPCML in inhibiting theproliferation of cancer cells in vitro. This can be determined usingmethods well known in the art and described in the Examples below.

In an embodiment, OPCML activity may be measured by the ability (e.g.,of the fragment or variant) to downregulate HER2, HER4, FGFR1, EPHA2and/or FGFR3. The activity of OPCML may also be defined by downstreamfunctional readouts such as phosphor ERK inactivation or downregulation,and also downregulation of phospho AKT.

The terms “nucleic acid molecule” and “polynucleotide” may be usedinterchangeably, and refer to a polymer of nucleotides. Such polymers ofnucleotides may contain natural and/or non-natural nucleotides, andinclude, but are not limited to, DNA, RNA, and PNA.

The terms “polypeptide” and “protein” are used interchangeably, andrefer to a polymer of amino acid residues. Except when the contextrequires otherwise, such polymers of amino acid residues may containnatural and/or non-natural amino acid residues. The terms “polypeptide”and “protein” also include post-translationally modified polypeptidesand proteins, including, for example, glycosylated, sialylated,acetylated, and/or phosphorylated polypeptides and proteins.

The OPCML, fragment or variant thereof may be prepared using an in vivoor in vitro expression system. Preferably, an expression system is usedthat provides the polypeptides in a form that is suitable forpharmaceutical use, and such expression systems are known to the skilledperson. As is clear to the skilled person, polypeptides of the inventionsuitable for pharmaceutical use can be prepared using techniques forpeptide synthesis.

A nucleic acid molecule encoding the OPCML, fragment or variant thereof,or fusion polypeptide thereof, may be used to transform a host cell orhost organism for expression of the desired polypeptide. Suitable hostsor host cells are known to the skilled person, and may be any suitablefungal, prokaryotic or eukaryotic cell or cell line or any suitablefungal, prokaryotic or eukaryotic organism, for example: a bacterialstrain, including but not limited to gram-negative strains such asstrains of Escherichia coli; of Proteus, for example of Proteusmirabilis; of Pseudomonas, for example of Pseudomonas fluorescens; andgram-positive strains such as strains of Bacillus, for example ofBacillus subtilis or of Bacillus brevis; of Streptomyces, for example ofStreptomyces lividans; of Staphylococcus, for example of Staphylococcuscarnosus; and of Lactococcus, for example of Lactococcus lactis; afungal cell, including but not limited to cells from species ofTrichoderma, for example from Trichoderma reesei; of Neurospora, forexample from Neurospora crassa; of Sordaria, for example from Sordariamacrospora; of Aspergillus, for example from Aspergillus niger or fromAspergillus sojae; or from other filamentous fungi; a yeast cell,including but not limited to cells from species of Saccharomyces, forexample of Saccharomyces cerevisiae; of Schizosaccharomyces, for exampleof Schizosaccharomyces pombe; of Pichia, for example of Pichia pastorisor of Pichia methanolica; of Hansenula, for example of Hansenulapolymorpha; of Kluyveromyces, for example of Kluyveromyces lactis; ofArxula, for example of Arxula adeninivorans; of Yarrowia, for example ofYarrowia lipolytica; an amphibian cell or cell line, such as Xenopusoocytes; an insect-derived cell or cell line, such as cells/cell linesderived from lepidoptera, including but not limited to Spodoptera SF9and Sf21 cells or cells/cell lines derived from Drosophila, such asSchneider and Kc cells; a plant or plant cell, for example in tobaccoplants; and/or a mammalian cell or cell line, for example a cell or cellline derived from a human, a cell or a cell line from mammals includingbut not limited to CHO-cells, BHK-cells (for example BHK-21 cells) andhuman cells or cell lines such as HeLa, COS (for example COS-7) andPER.C6® cells; as well as all other hosts or host cells known per se forthe expression and production of polypeptides known to the skilledperson.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of OPCML, a fragment or variant thereof, or apolypeptide fusion thereof, include strains of E. coli, Pichia pastorisand S. cerevisiae that are suitable for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical (i.e. GMP grade) expression/production/fermentation.Suitable examples of such strains are known to the skilled person. Suchstrains and production/expression systems are commercially available bycompanies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are commercially available.

The choice of the specific expression system depends, in part, on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a protein for whichglycosylation is desired or required necessitates the use of mammalianexpression hosts that have the ability to glycosylate the expressedprotein. In this respect, it is clear to the skilled person that theglycosylation pattern obtained (i.e. the kind, number and position ofresidues attached) will depend on the cell or cell line that is used forthe expression. Preferably, either a human cell or cell line is used(i.e. leading to a protein that essentially has a human glycosylationpattern) or another mammalian cell line is used that can provide aglycosylation pattern that is essentially and/or functionally the sameas human glycosylation or at least mimics human glycosylation.Generally, prokaryotic hosts such as E. coli do not have the ability toglycosylate proteins, and the use of lower eukaryotes such as yeastusually leads to a glycosylation pattern that differs from humanglycosylation. Nevertheless, it should be understood that all thedescribed host cells and expression systems may be used in theinvention, depending on the desired polypeptide amino acid sequence tobe obtained and its desired use. Thus, according to one embodiment, theOPCML, fragment or variant thereof, or polypeptide fusion thereof, isglycosylated. According to an alternative embodiment, it may not beglycosylated.

Thus the OPCML, fragment or variant thereof, or a polypeptide fusionthereof (see below), may be produced in a bacterial cell, in a yeastcell, in an insect cell, in a plant cell, or in a mammalian cell, inparticular in a human cell or in a cell of a human cell line, which issuitable for large scale pharmaceutical production.

When expression in a host cell is used to produce the OPCML, fragment orvariant thereof, or polypeptide fusion thereof, they can be producedeither intracellullarly (e.g. in the cytosol, in the periplasm or ininclusion bodies) and then isolated from the host cells and optionallyfurther purified; or they can be produced extracellularly (e.g. in themedium in which the host cells are cultured) and then isolated from theculture medium and optionally further purified. When eukaryotic hostcells are used, extracellular production is usually preferred since thisconsiderably facilitates further isolation and downstream processing.

Bacterial cells such as strains of E. coli normally do not secreteproteins extracellularly, except for a few classes of proteins such astoxins and hemolysin, and secretory production in E. coli refers to thetranslocation of proteins across the inner membrane to the periplasmicspace. Periplasmic production provides several advantages over cytosolicproduction. For example, the N-terminal amino acid sequence of thesecreted product can be identical to a natural gene product aftercleavage of the secretion signal sequence by a specific signalpeptidase. Also, there appears to be much less protease activity in theperiplasm than in the cytoplasm. In addition, protein purification issimpler due to fewer contaminating proteins in the periplasm. Anotheradvantage is that correct disulfide bonds may form because the periplasmprovides a more oxidative environment than the cytoplasm. Proteinsoverexpressed in E. coli are often found in insoluble aggregates,so-called inclusion bodies. These inclusion bodies may be located in thecytosol or in the periplasm; the recovery of biologically activeproteins from these inclusion bodies requires a denaturation/refoldingprocess. Many recombinant proteins, including therapeutic proteins, arerecovered from inclusion bodies. Alternatively, recombinant strains ofbacteria that have been genetically modified so as to secrete a desiredprotein can be used.

Some preferred promoters for use with host cells include, for expressionin E. coli: lac promoter (and derivatives thereof such as the lacUV5promoter); arabinose promoter; left-(PL) and rightward (PR) promoter ofphage lambda; promoter of the trp operon; hybrid lac/trp promoters (tacand trc); T7-promoter (more specifically that of T7-phage gene 10) andother T-phage promoters; promoter of the Tn10 tetracycline resistancegene; engineered variants of the above promoters that include one ormore copies of an extraneous regulatory operator sequence; forexpression in S. cerevisiae: constitutive: ADH1 (alcohol dehydrogenase1), ENO (enolase), CYC1 (cytochrome c iso-1), GAPDH(glyceraldehydes-3-phosphate dehydrogenase), PGK1 (phosphoglyceratekinase), PYK1 (pyruvate kinase); regulated: GAL1, 10, 7 (galactosemetabolic enzymes), ADH2 (alcohol dehydrogenase 2), PHO5 (acidphosphatase), CUP1 (copper metallothionein); heterologous: CaMV(cauliflower mosaic virus 35S promoter); for expression in Pichiapastoris: the AOX1 promoter (alcohol oxidase I); for expression inmammalian cells: human cytomegalovirus (hCMV) immediate earlyenhancer/promoter; human cytomegalovirus (hCMV) immediate early promotervariant that contains two tetracycline operator sequences such that thepromoter can be regulated by the Tet repressor; Herpes Simplex Virusthymidine kinase (TK) promoter; Rous Sarcoma Virus long terminal repeat(RSV LTR) enhancer/promoter; elongation factor 1α (hEF-1α) promoter fromhuman, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1 longterminal repeat promoter; and the β-actin promoter.

Some preferred vectors for use with host cells include: vectors forexpression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen),pMC1neo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593),pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt(ATCC37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC37460) and 1ZD35 (ATCC 37565), as well as viral-based expressionsystems, such as those based on adenovirus; vectors for expression inbacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen); vectorsfor expression in yeast or other fungal cells: pYES2 (Invitrogen) andPichia expression vectors (Invitrogen); vectors for expression in insectcells: pBlueBacII (Invitrogen) and other baculovirus vectors; vectorsfor expression in plants or plant cells: for example vectors based oncauliflower mosaic virus or tobacco mosaic virus, suitable strains ofAgrobacterium, or Ti-plasmid based vectors.

Some preferred secretory sequences for use with these host cellsinclude: for use in bacterial cells such as E. coli: PeIB, Bla, OmpA,OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the like; TATsignal peptide, hemolysin C-terminal secretion signal; for use in yeast:α-mating factor prepro-sequence, phosphatase (phol), invertase (Suc),etc.; for use in mammalian cells: indigenous signal in case the targetprotein is of eukaryotic origin; murine Ig κ-chain V-J2-C signalpeptide; etc.

Suitable techniques for transforming a host or host cell of theinvention are well known to the skilled person and depend on theintended host cell/host organism and the genetic construct to be used.

After transformation a step for detecting and selecting those host cellsor host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct or a step involving the detection ofthe OPCML amino acid sequence, e.g. using specific antibodies.

Preferably, the host cells express, or are (at least) capable ofexpressing (e.g. under suitable conditions), the OPCML, fragment orvariant thereof, or polypeptide fusion thereof. To produce/obtainexpression of the OPCML, fragment or variant thereof, or polypeptidefusion thereof, the transformed host cell may generally be kept,maintained and/or cultured under conditions such that the polypeptide isexpressed/produced. Suitable conditions are known to the skilled personand depend upon the host cell/host organism used, as well as on theregulatory elements that control the expression of the (relevant)nucleotide sequence.

Generally, suitable conditions include the use of a suitable medium, thepresence of a suitable source of food and/or suitable nutrients, the useof a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences areunder the control of an inducible promoter); all of which may beselected by the skilled person. Again, under such conditions, thedesired polypeptide may be expressed in a constitutive manner, in atransient manner, or only when suitably induced.

It will also be clear to the skilled person that the OPCML, fragment orvariant thereof, or polypeptide fusion thereof, may (first) be generatedin an immature form, which may then be subjected to post-translationalmodification, depending on the host cell/host organism used. Also, theOPCML, fragment or variant thereof, or polypeptide fusion thereof, maybe glycosylated, again depending on the host cell/host organism used.

The OPCML, fragment or variant thereof, or polypeptide fusion thereof,may then be isolated from the host cell/host organism and/or from themedium in which it was cultivated, using protein isolation and/orpurification techniques known per se, such as (preparative)chromatography and/or electrophoresis techniques, differentialprecipitation techniques, affinity techniques (e.g. using a specific,cleavable amino acid sequence fused with the OPCML, fragment or variantthereof, or polypeptide fusion thereof) and/or preparative immunologicaltechniques (i.e. using antibodies against the polypeptide to beisolated).

Less preferably, the OPCML, fragment or variant thereof, may be made bychemical synthesis. For example, peptides may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Luet al (1981) J. Org. Chem. 46, 3433 and references therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is effected using 20% piperidine in N,N-dimethylformamide.Side-chain functionalities may be protected as their butyl ethers (inthe case of serine threonine and tyrosine), butyl esters (in the case ofglutamic acid and aspartic acid), butyloxycarbonyl derivative (in thecase of lysine and histidine), trityl derivative (in the case ofcysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (inthe case of arginine). Where glutamine or asparagine are C-terminalresidues, use is made of the 4,4′-dimethoxybenzhydryl group forprotection of the side chain amido functionalities. The solid-phasesupport is based on a polydimethyl-acrylamide polymer constituted fromthe three monomers dimethylacrylamide (backbone-monomer),bisacryloylethylene diamine (cross linker) and acryloylsarcosine methylester (functionalizing agent). The peptide-to-resin cleavable linkedagent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acidderivative. All amino acid derivatives are added as their preformedsymmetrical anhydride derivatives with the exception of asparagine andglutamine, which are added using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesized. Trifluoroacetic acid is removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present are removed by a simple extractionprocedure which on lyophilization of the aqueous phase affords the crudepeptide free of scavengers. Reagents for peptide synthesis are generallyavailable from Calbiochem-Novabiochem (UK). Purification may be effectedby any one, or a combination of, techniques such as size exclusionchromatography, ion-exchange chromatography and (principally)reverse-phase high performance liquid chromatography. Analysis ofpeptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis.

Nucleic acid molecules encoding the OPCML, fragment or variant thereof,or a polypeptide fusion thereof, may be prepared using methods very wellknown in the art of molecular biology. For example, many of thetechniques used in connection with recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation (e.g., electroporation,lipofection), enzymatic reactions, and purification techniques are knownin the art. Many such techniques and procedures are described, e.g., inSambrook et al. Molecular Cloning: A Laboratory Manual (2^(nd) edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)),among other places.

In certain embodiments, the OPCML, fragment or variant thereof may be inthe form of a fusion molecule in which the OPCML, fragment or variantthereof is attached to a fusion partner to form a fusion protein. Manydifferent types of fusion partners are known in the art. One skilled inthe art can select a suitable fusion partner according to the intendeduse of the fusion protein. Examples of fusion partners include polymers,polypeptides, lipophilic moieties, and succinyl groups. Preferredprotein fusion partners include serum albumin and an antibody Fc domain.Preferred polymer fusion partners include, but are not limited to,polyethylene glycol, including polyethylene glycols having branchedand/or linear chains. Thus, in certain preferred embodiments, the OPCMLpolypeptide or fragment or variant thereof may be PEGylated, or maycomprise a fusion protein with an Fc fragment.

In various embodiments, oligomerization may offer certain functionaladvantages to a fusion protein, including, but not limited to,multivalency, increased binding strength, and the combined function ofdifferent domains. Accordingly, in certain embodiments, a fusion partnercomprises an oligomerization domain, for example, a dimerization domain.Exemplary oligomerization domains include, but are not limited to,coiled-coil domains, including alpha-helical coiled-coil domains;collagen domains; collagen-like domains, and certain immunoglobulindomains. Certain exemplary coiled-coil polypeptide fusion partnersinclude the tetranectin coiled-coil domain; the coiled-coil domain ofcartilage oligomeric matrix protein; angiopoietin coiled-coil domains;and leucine zipper domains. Certain exemplary collagen or collagen-likeoligomerization domains include, but are not limited to, those found incollagens, mannose binding lectin, lung surfactant proteins A and D,adiponectin, ficolin, conglutinin, macrophage scavenger receptor, andemilin.

In certain embodiments, the fusion partner may be an albumin. Certainexemplary albumins include, but are not limited to, human serum album(HSA) and fragments of HSA that are capable of increasing the serumhalf-life and/or bioavailability of the polypeptide to which they arefused. In certain embodiments, a fusion partner is an albumin-bindingmolecule, such as, for example, a peptide that binds albumin or amolecule that conjugates with a lipid or other molecule that bindsalbumin. In certain embodiments, a fusion molecule comprising HSA isprepared as described, e.g., in U.S. Pat. No. 6,686,179.

Proteins can be linked to an Fc region of an IgG molecule. The Fc regionof an IgG molecule refers to the Fc domain of an immunoglobulin of theisotype IgG, as is well known to those skilled in the art. The Fc regionof an IgG molecule is that portion of IgG molecule (IgG1, IgG2, IgG3,and IgG4) that is responsible for increasing the in vivo serum half-lifeof the IgG molecule, typically by avoiding rapid renal clearance.Proteins may be fused adjacent to the Fc region of the IgG molecule, orattached to the Fc region of the IgG molecule via a linker peptide. Useof such linker peptides is well known in protein biochemistry. The Fcregion is typically derived from IgG1 or IgG4. An example of a suitableFc domain is the Fc domain derived from human IgG1 that is contained inthe plasmid pFUSE-hlgG1e1-Fc1 (InvivoGen).

Therapeutic proteins produced as an Fc-chimera are known in the art. Forexample, Etanercept, the extracellular domain of TNFR2 combined with anFc fragment, is a therapeutic polypeptide used to treat autoimmunediseases, such as rheumatoid arthritis.

Fusion to an Fc domain may also induce the formation of a dimericspecies. Since we have shown that recombinant OPCML produced in bacteriaforms a dimer, this may be useful in certain embodiments. Fc-fusions mayalso improve efficacy of a therapeutic polypeptide through enhancedstability and by increasing the molecular weight of the molecule, thusreducing the rate of renal clearance.

When the fusion partner is an Fc immunoglobulin domain, the skilledperson can select an appropriate Fc domain fusion partner according tothe intended use. An Fc fusion partner may be a wild-type Fc found in anaturally occurring antibody, a variant thereof, or a fragment thereof.Non-limiting exemplary Fc fusion partners include Fcs comprising a hingeand the CH2 and CH3 constant domains of a human IgG, for example, humanIgG1, IgG2, IgG3, or IgG4. Certain additional Fc fusion partnersinclude, but are not limited to, human IgA and IgM. In certainembodiments, an Fc fusion partner comprises a C237S mutation. In certainembodiments, an Fc fusion partner comprises a hinge, CH2, and CH3domains of human IgG2 with a P331S mutation, as described in U.S. Pat.No. 6,900,292.

In certain embodiments, the fusion partner may be a polymer, forexample, polyethylene glycol (PEG). PEG may comprise branched and/orlinear chains. In certain embodiments, a fusion partner comprises achemically-derivatized polypeptide having at least one PEG moietyattached.

Pegylation is a process whereby polyethylene glycol (PEG) is attached toa protein in order to extend the half-life of the protein in the body.Pegylation of proteins may decrease the dose or frequency ofadministration of the proteins needed for an optimal activity. (Reviewsof the technique are provided in Bhadra et al (2002) Pharmazie 57: 5-29and in Harris et al (2001) Clin. Pharmacokinet, 40: 539-551.)

Pegylation of a polypeptide may be carried out by any method known inthe art. One skilled in the art can select an appropriate method ofpegylating a particular polypeptide, taking into consideration theintended use of the polypeptide. Certain exemplary PEG attachmentmethods as described, for example, in Malik et al., (1992) Exp.Hematol., 20: 1028-1035; Francis (1992) Focus on Growth Factors 3: 4-10;EP 0 401 384; EP 0 154 316; EP 0 401 384; WO 92/16221; and WO 95/34326.Pegylation may be performed via an acylation reaction or an alkylationreaction, resulting in attachment of one or more PEG moieties via acylor alkyl groups. In certain embodiments, PEG moieties are attached to apolypeptide through the α- or ε-amino group of one or more amino acids,although any other points of attachment known in the art are alsocontemplated.

Pegylation by acylation typically involves reacting an activated esterderivative of a PEG moiety with a polypeptide. An example of activatedPEG ester is PEG esterified to N-hydroxysuccinimide (NHS). As usedherein, acylation is contemplated to include, without limitation, thefollowing types of linkages between a polypeptide and PEG: amide,carbamate, and urethane. See, e.g., Chamow, (1994) Bioconjugate Chem.,5: 133-140. Pegylation by alkylation typically involves reacting aterminal aldehyde derivative of a PEG moiety with a polypeptide in thepresence of a reducing agent. Non-limiting exemplary reactive PEGaldehydes include PEG propionaldehyde, which is water stable, and monoC1-C10 alkoxy or aryloxy derivatives thereof (See U.S. Pat. No.5,252,714).

In certain embodiments, a pegylation reaction results in poly-pegylatedpolypeptides. In certain embodiments, a pegylation reaction results inmono-, di-, and/or tri-pegylated polypeptides. One skilled in the artcan select appropriate pegylation chemistry and reaction conditions toachieve the desired level of pegylation. Further, desired pegylatedspecies may be separated from a mixture containing other pegylatedspecies and/or unreacted starting materials using various purificationtechniques known in the art, including among others, dialysis,salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography, and electrophoresis.

The fusion partner may be attached, either covalently or non-covalently,to the amino-terminus or the carboxy-terminus of the OPCML, fragment orvariant thereof. The attachment may also occur at a location within theOPCML, fragment or variant thereof other than the amino-terminus or thecarboxy-terminus, for example, through an amino acid side chain (suchas, for example, the side chain of cysteine, lysine, histidine, serine,or threonine).

In either covalent or non-covalent attachment embodiments, a linker maybe included between the fusion partner and the OPCML, fragment orvariant thereof. Such linkers may be comprised of amino acids and/orchemical moieties. One skilled in the art can select a suitable linkerdepending on the attachment method used, the intended use of the OPCML,fragment or variant thereof, and the desired spacing between the OPCML,fragment or variant thereof and the fusion partner.

Exemplary methods of covalently attaching a fusion partner to apolypeptide include, but are not limited to, translation of thepolypeptide and the fusion partner as a single amino acid sequence, andchemical attachment of the fusion partner to the polypeptide. When thefusion partner and the polypeptide are translated as single amino acidsequence, additional amino acids may be included between the fusionpartner and the polypeptide as a linker. In certain embodiments, thelinker is glycine-serine (“GS”). In certain embodiments, the linker isselected based on the polynucleotide sequence that encodes it, tofacilitate cloning the fusion partner and the polypeptide into a singleexpression construct (for example, a polynucleotide containing aparticular restriction site may be placed between the polynucleotideencoding the fusion partner and the polynucleotide encoding the OPCML,fragment or variant thereof, wherein the polynucleotide containing therestriction site encodes a short amino acid linker sequence).

When the fusion partner and the OPCML, fragment or variant thereof arecovalently coupled by chemical means, linkers of various sizes cantypically be included during the coupling reaction. One skilled in theart can select a suitable method of covalently attaching a fusionpartner to a polypeptide depending, for example, on the identity of thefusion partner and the particular use intended for the fusion molecule.One skilled in the art can also select a suitable linker type andlength, if one is desired.

Exemplary methods of non-covalently attaching a fusion partner to apolypeptide include, but are not limited to, attachment through abinding pair. Exemplary binding pairs include, but are not limited to,biotin and avidin or streptavidin, an antibody and its antigen, etc.Again, one skilled in the art can select a suitable method ofnon-covalently attaching a fusion partner to a polypeptide depending,for example, on the identity of the fusion partner and the particularuse intended for the fusion molecule. The selected non-covalentattachment method should be suitable for the conditions under which thefusion molecule will be used, taking into account, for example, the pH,salt concentrations, and temperature.

It is appreciated that the compound comprising or consisting of theOPCML polypeptide or fragment or variant thereof, of the fusion thereof,or nucleic acid molecule encoding the OPCML polypeptide or fragment orvariant, or polypeptide fusion thereof, may be formulated as ananoparticle. Nanoparticles are a colloidal carrier system that has beenshown to improve the efficacy of an encapsulated drug by prolonging theserum half-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are apolymer colloidal drug delivery system that is in clinical development(described, for example, by Stella et al (2000) J. Pharm. Sci., 89:1452-1464; Brigger et al (2001) Int. J. Pharm 214: 37-42; Calvo et al(2001) Pharm. Res. 18: 1157-1166; and Li et al (2001) Biol. Pharm. Bull.24: 662-665). Biodegradable poly(hydroxyl acids), such as the copolymersof poly(lactic acid) (PLA) and poly(lactic-co-glycolide) (PLGA) arebeing extensively used in biomedical applications and have received FDAapproval for certain clinical applications. In addition, PEG-PLGAnanoparticles have many desirable carrier features including (i) thatthe agent to be encapsulated comprises a reasonably high weight fraction(loading) of the total carrier system; (ii) that the amount of agentused in the first step of the encapsulation process is incorporated intothe final carrier (entrapment efficiency) at a reasonably high level;(iii) that the carrier has the ability to be freeze-dried andreconstituted in solution without aggregation; (iv) that the carrier bebiodegradable; (v) that the carrier system be of small size; and (vi)that the carrier enhances the particles persistence.

Nanoparticles may be synthesized using virtually any biodegradable shellknown in the art. In one embodiment, a polymer, such aspoly(lactic-acid) (PLA) or poly(lactic-co-glycolic acid) (PLGA) is used.Such polymers are biocompatible and biodegradable, and are subject tomodifications that desirably increase the photochemical efficacy andcirculation lifetime of the nanoparticle. In one embodiment, the polymeris modified with a terminal carboxylic acid group (COOH) that increasesthe negative charge of the particle and thus limits the interaction withnegatively charged nucleic acids. Nanoparticles may also be modifiedwith polyethylene glycol (PEG), which also increases the half-life andstability of the particles in circulation. Alternatively, the COOH groupmay be converted to an N-hydroxysuccinimide (NHS) ester for covalentconjugation to amine-modified compounds.

Other protein modifications to stabilize a polypeptide, for example toprevent degradation, as are well known in the art may also be employed.Specific amino acids may be modified to reduce cleavage of thepolypeptide in vivo; typically, N- or C-terminal regions are modified toreduce protease activity on the polypeptide. A stabilizing modificationis any modification known in the art or described herein capable ofstabilizing a protein, enhancing the in vitro half life of a protein,enhancing circulatory half life of a protein and/or reducing proteolyticdegradation of a protein. For example, polypeptides may be linked to theserum albumin or a derivative of albumin. Methods for linkingpolypeptides to albumin or albumin derivatives are well known in the art(e.g., U.S. Pat. No. 5,116,944).

It is appreciated that the compound comprising or consisting of theOPCML polypeptide or fragment or variant thereof, or nucleic acidmolecule encoding the OPCML polypeptide or fragment or variant, willtypically be formulated for administration to an individual as apharmaceutical composition, i.e. together with a pharmaceuticallyacceptable carrier, diluent or excipient.

By “pharmaceutically acceptable” is included that the formulation issterile and pyrogen free. Suitable pharmaceutical carriers, diluents andexcipients are well known in the art of pharmacy. The carrier(s) must be“acceptable” in the sense of being compatible with the compound and notdeleterious to the recipients thereof. Typically, the carriers will bewater or saline which will be sterile and pyrogen free; however, otheracceptable carriers may be used.

In an embodiment, the pharmaceutical compositions or formulations of theinvention are formulated for parenteral administration, moreparticularly for intravenous administration. In a preferred embodiment,the pharmaceutical composition is suitable for intravenousadministration to a patient, for example by injection.

Thus, typically and preferably, the compound is administeredintravenously or by intraperitoneal administration to the patient.

Suitably and equally preferably, the compound is administered as aninfusion or as a bolus injection.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

In an alternative preferred embodiment, the pharmaceutical compositionis suitable for topical administration to a patient.

Preferably, the formulation is a unit dosage containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of the activeingredient.

The compound may be administered orally or by any parenteral route, inthe form of a pharmaceutical formulation comprising the activeingredient, optionally in the form of a non-toxic organic, or inorganic,acid, or base, addition salt, in a pharmaceutically acceptable dosageform. Depending upon the disorder and patient to be treated, as well asthe route of administration, the compositions may be administered atvarying doses.

We have tested 0.5, 1, 2, 5 and 10 μM of recombinant full length humanOPCML in vitro, and there is a linear dose response which has not yetpeaked. We see inhibition of cell growth from 2 μm and frank cell deathat 5-10 μM in 6/7 ovarian cancer cell lines and 2/2 breast cancer celllines (both HER2 positive and HER2 negative). The physician skilled inthe art will be able to identify suitable doses based upon thisinformation and their skill in the art.

In human therapy, the compound will generally be administered inadmixture with a suitable pharmaceutical excipient, diluent or carrierselected with regard to the intended route of administration andstandard pharmaceutical practice.

For example, the compound may be administered orally, buccally orsublingually in the form of tablets, capsules, ovules, elixirs,solutions or suspensions, which may contain flavouring or colouringagents, for immediate-, delayed- or controlled-release applications. Thecompound may also be administered via intracavernosal injection.

Suitable tablets may contain excipients such as microcrystallinecellulose, lactose, sodium citrate, calcium carbonate, dibasic calciumphosphate and glycine, disintegrants such as starch (preferably corn,potato or tapioca starch), sodium starch glycolate, croscarmellosesodium and certain complex silicates, and granulation binders such aspolyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC),hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia.Additionally, lubricating agents such as magnesium stearate, stearicacid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

The compound can also be administered parenterally, for example,intravenously, intra-arterially, intraperitoneally, intrathecally,intraventricularly, intrasternally, intracranially, intra-muscularly orsubcutaneously, or they may be administered by infusion techniques. Theyare best used in the form of a sterile aqueous solution which maycontain other substances, for example, enough salts or glucose to makethe solution isotonic with blood. The aqueous solutions should besuitably buffered (preferably to a pH of from 3 to 9), if necessary. Thepreparation of suitable parenteral formulations under sterile conditionsis readily accomplished by standard pharmaceutical techniques well-knownto those skilled in the art.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilised) condition requiring only the addition of thesterile liquid carrier, for example water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

For oral and parenteral administration to human patients, the dailydosage level of a compound will usually be from 1 to 1,000 mg per adult(i.e. from about 0.015 to 15 mg/kg), administered in single or divideddoses.

Thus, for example, the tablets or capsules of the compound may containfrom 1 mg to 1,000 mg of active agent for administration singly or twoor more at a time, as appropriate. The physician in any event willdetermine the actual dosage which will be most suitable for anyindividual patient and it will vary with the age, weight and response ofthe particular patient. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited and such are within the scope of thisinvention.

The compound can also be administered intranasally or by inhalation andare conveniently delivered in the form of a dry powder inhaler or anaerosol spray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, e.g.dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas. In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activecompound, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of an antibody and a suitable powder base such as lactose or starch.Such formulations may be particularly useful for treating solid tumoursof the lung.

Aerosol or dry powder formulations are preferably arranged so that eachmetered dose or “puff” contains at least 1 mg of the compound fordelivery to the patient. It will be appreciated that the overall dailydose with an aerosol will vary from patient to patient, and may beadministered in a single dose or, more usually, in divided dosesthroughout the day.

Alternatively, the compound can be administered in the form of asuppository or pessary, particularly for treating or targeting colon,rectal or prostate tumours.

The compound may also be administered by the ocular route. Forophthalmic use, the compound can be formulated as, e.g., micronisedsuspensions in isotonic, pH adjusted, sterile saline, or, preferably, assolutions in isotonic, pH adjusted, sterile saline, optionally incombination with a preservative such as a benzylalkonium chloride.Alternatively, they may be formulated in an ointment such as petrolatum.Such formulations may be particularly useful for treating cancers of theeye, such as retinoblastoma.

The compound may be applied topically in the form of a lotion, solution,cream, ointment or dusting powder, or may be transdermally administered,for example, by the use of a skin patch. For application topically tothe skin, the compound can be formulated as a suitable ointmentcontaining the active compound suspended or dissolved in, for example, amixture with one or more of the following: mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylenepolyoxypropylene compound, emulsifying wax and water. Alternatively,they can be formulated as a suitable lotion or cream, suspended ordissolved in, for example, a mixture of one or more of the following:mineral oil, sorbitan monostearate, a polyethylene glycol, liquidparaffin, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. Such formulations may beparticularly useful for treating skin cancers.

For skin cancers, the compound can also be delivered byelectroincorporation (EI). EI occurs when small particles of up to 30microns in diameter on the surface of the skin experience electricalpulses identical or similar to those used in electroporation. In EI,these particles are driven through the stratum corneum and into deeperlayers of the skin. The particles can be loaded or coated with thecompound or can simply act as “bullets” that generate pores in the skinthrough which the compound can enter.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier. Such formulations may be particularly usefulfor treating cancers of the mouth and throat.

In an embodiment, when the compound is a polypeptide, it may bedelivered using an injectable sustained-release drug delivery system.These are designed specifically to reduce the frequency of injections.An example of such a system is Nutropin Depot which encapsulatesrecombinant human growth hormone (rhGH) in biodegradable microspheresthat, once injected, release rhGH slowly over a sustained period.

The antibody can be administered by a surgically implanted device thatreleases the drug directly to the required site, for example, into theeye to treat ocular tumours. Such direct application to the site ofdisease achieves effective therapy without significant systemicside-effects.

An alternative method for delivery of polypeptides is the ReGelinjectable system that is thermo-sensitive. Below body temperature,ReGel is an injectable liquid while at body temperature it immediatelyforms a gel reservoir that slowly erodes and dissolves into known, safe,biodegradable polymers. The active drug is delivered over time as thebiopolymers dissolve.

Polypeptides can also be delivered orally. For example, the processemploys a natural process for oral uptake of vitamin B₁₂ in the body toco-deliver proteins and peptides. By riding the vitamin B₁₂ uptakesystem, the protein or peptide can move through the intestinal wall.Complexes are synthesised between vitamin B₁₂ analogues and the drugthat retain both significant affinity for intrinsic factor (IF) in thevitamin B₁₂ portion of the complex and significant bioactivity of thedrug portion of the complex.

Polynucleotides may be administered by any effective method, forexample, parenterally (e.g. intravenously, subcutaneously,intramuscularly) or by oral, nasal or other means which permit thepolynucleotides to access and circulate in the patient's bloodstream.Polynucleotides administered systemically preferably are given inaddition to locally administered polynucleotides, but also have utilityin the absence of local administration. A dosage in the range of fromabout 0.1 to about 10 grams per administration to an adult humangenerally will be effective for this purpose.

The polynucleotide may be administered as a suitable genetic constructas is described below and delivered to the patient where it isexpressed. Typically, the polynucleotide in the genetic construct isoperatively linked to a promoter which can express the compound in thecell. The genetic constructs of the invention can be prepared usingmethods well known in the art, for example in Sambrook et al (2001).

Although genetic constructs for delivery of polynucleotides can be DNAor RNA, it is preferred if they are DNA.

Preferably, the genetic construct is adapted for delivery to a humancell.

Means and methods of introducing a genetic construct into a cell in ananimal body are known in the art. For example, the constructs of theinvention may be introduced into cells by any convenient method, forexample methods involving retroviruses, so that the construct isinserted into the genome of the cell. For example, in Kuriyama et al(1991, Cell Struc. and Func. 16, 503-510), purified retroviruses areadministered. Retroviral DNA constructs comprising a polynucleotide asdescribed above may be made using methods well known in the art. Toproduce active retrovirus from such a construct it is usual to use anecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% foetal calf serum (FCS). Transfection ofthe cell line is conveniently by calcium phosphate co-precipitation, andstable transformants are selected by addition of G418 to a finalconcentration of 1 mg/ml (assuming the retroviral construct contains aneo^(R) gene). Independent colonies are isolated and expanded and theculture supernatant removed, filtered through a 0.45 μm pore-size filterand stored at −70° C. For the introduction of the retrovirus into tumourcells, for example, it is convenient to inject directly retroviralsupernatant to which 10 μg/ml Polybrene has been added. For tumoursexceeding 10 mm in diameter it is appropriate to inject between 0.1 mland 1 ml of retroviral supernatant; preferably 0.5 Al.

Alternatively, as described in Culver et al (1992, Science 256,1550-1552), cells which produce retroviruses may be injected. Theretrovirus-producing cells so introduced are engineered to activelyproduce retroviral vector particles so that continuous production of thevector occurs within the tumour mass in situ.

Targeted retroviruses are also available for use in the invention; forexample, sequences conferring specific binding affinities may beengineered into pre-existing viral env genes (see Miller & Vile (1995)Faseb J. 9, 190-199, for a review of this and other targeted vectors forgene therapy).

Other methods involve simple delivery of the construct into the cell forexpression therein either for a limited time or, following integrationinto the genome, for a longer time. An example of the latter approachincludes liposomes (Nässander et al (1992) Cancer Res. 52, 646-653).

Other methods of delivery include adenoviruses carrying external DNA viaan antibody-polylysine bridge (see Curiel (1993) Prog. Med. Virol. 40,1-18) and transferrin-polycation conjugates as carriers (Wagner et al(1990) Proc. Natl. Acad. Sol. USA 87, 3410-3414). In the first of thesemethods a polycation-antibody complex is formed with the DNA constructor other genetic construct of the invention, wherein the antibody isspecific for either wild-type adenovirus or a variant adenovirus inwhich a new epitope has been introduced which binds the antibody. Thepolycation moiety binds the DNA via electrostatic interactions with thephosphate backbone. The adenovirus, because it contains unaltered fibreand penton proteins, is internalised into the cell and carries into thecell with it the DNA construct of the invention. It is preferred if thepolycation is polylysine.

In an alternative method, a high-efficiency nucleic acid delivery systemthat uses receptor-mediated endocytosis to carry DNA macromolecules intocells is employed. This is accomplished by conjugating theiron-transport protein transferrin to polycations that bind nucleicacids. Human transferrin, or the chicken homologue conalbumin, orcombinations thereof is covalently linked to the small DNA-bindingprotein protamine or to polylysines of various sizes through adisulphide linkage. These modified transferrin molecules maintain theirability to bind their cognate receptor and to mediate efficient irontransport into the cell. The transferrin-polycation molecules formelectrophoretically stable complexes with DNA constructs or othergenetic constructs of the invention independent of nucleic acid size(from short oligonucleotides to DNA of 21 kilobase pairs). Whencomplexes of transferrin-polycation and the DNA constructs or othergenetic constructs of the invention are supplied to the tumour cells, ahigh level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs orother genetic constructs of the invention using the endosome-disruptionactivity of defective or chemically inactivated adenovirus particlesproduced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci.USA 89, 6094-6098 may also be used. This approach appears to rely on thefact that adenoviruses are adapted to allow release of their DNA from anendosome without passage through the lysosome, and in the presence of,for example transferrin linked to the DNA construct or other geneticconstruct of the invention, the construct is taken up by the cell by thesame route as the adenovirus particle. This approach has the advantagesthat there is no need to use complex retroviral constructs; there is nopermanent modification of the genome as occurs with retroviralinfection; and the targeted expression system is coupled with a targeteddelivery system, thus reducing toxicity to other cell types.

It will be appreciated that “naked DNA” and DNA complexed with cationicand neutral lipids may also be useful in introducing the DNA of theinvention into cells of the individual to be treated. Non-viralapproaches to gene therapy are described in Ledley (1995, Human GeneTherapy 6, 1129-1144).

For cancers of specific tissues it may be useful to use tissue-specificpromoters in the vectors encoding a polynucleotide. This is because thetargeted genes are only expressed, or selectively expressed, in thetumour endothelium.

Targeted delivery systems are also known, such as the modifiedadenovirus system described in WO 94/10323, wherein, typically, the DNAis carried within the adenovirus, or adenovirus-like, particle. Michaelet al (1995) Gene Therapy 2: 660-668, describes modification ofadenovirus to add a cell-selective moiety into a fibre protein. Mutantadenoviruses which replicate selectively in p53-deficient human tumourcells, such as those described in Bischoff et al (1996) Science 274:373-376 are also useful for delivering genetic constructs to a cell.Other suitable viruses, viral vectors or virus-like particles includelentivirus and lentiviral vectors, HSV, adeno-assisted virus (AAV) andAAV-based vectors, vaccinia and parvovirus.

Methods of delivering polynucleotides to a patient are well known to aperson of skill in the art and include the use of immunoliposomes, viralvectors (including vaccinia, modified vaccinia, adenovirus andadeno-associated viral (AAV) vectors), and by direct delivery of DNA,e.g. using a gene-gun and electroporation. Furthermore, methods ofdelivering polynucleotides to a target tissue of a patient for treatmentare also well known in the art.

Methods of targeting and delivering therapeutic agents directly tospecific regions of the body are well known to a person of skill in theart.

For example, U.S. Pat. No. 6,503,242 describes an implanted catheterapparatus for delivering therapeutic agents directly to the hippocampus.Methods of targeting and delivering agents to the brain can be used forthe treatment of solid tumours of the brain. In one embodiment,therapeutic agents including vectors can be distributed throughout awide region of the CNS by injection into the cerebrospinal fluid, e.g.,by lumbar puncture (See e.g., Kapadia et al (1996) Neurosurg 10:585-587). Alternatively, precise delivery of the therapeutic agent intospecific sites of the brain can be conducted using stereotacticmicroinjection techniques. For example, the subject being treated can beplaced within a stereotactic frame base (MRI-compatible) and then imagedusing high resolution MRI to determine the three-dimensional positioningof the particular region to be treated. The MRI images can then betransferred to a computer having the appropriate stereotactic software,and a number of images are used to determine a target site andtrajectory for microinjection of the therapeutic agent. The softwaretranslates the trajectory into three-dimensional coordinates that areprecisely registered for the stereotactic frame. In the case ofintracranial delivery, the skull will be exposed, burr holes will bedrilled above the entry site, and the stereotactic apparatus used toposition the needle and ensure implantation at a predetermined depth.The therapeutic agent can be delivered to regions of the CNS such as thehippocampus, cells of the spinal cord, brainstem, (medulla, pons, andmidbrain), cerebellum, diencephalon (thalamus, hypothalamus),telencephalon (corpus stratium, cerebral cortex, or within the cortex,the occipital, temporal, parietal or frontal lobes), or combinations,thereof. In another embodiment, the therapeutic agent is delivered usingother delivery methods suitable for localised delivery, such aslocalised permeation of the blood-brain barrier. US 2005/0025746describes delivery systems for localised delivery of an adeno-associatedvirus vector (AAV) vector encoding a therapeutic agent to a specificregion of the brain.

When a therapeutic agent for the treatment of a solid cancer of, forexample, the brain, is encoded by a polynucleotide, it may be preferablefor its expression to be under the control of a suitable tissue-specificpromoter. Central nervous system (CNS) specific promoters such as,neuron-specific promoters (e.g., the neurofilament promoter (Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477) and glialspecific promoters (Morii et al (1991) Biochem. Biophys Res. Commun.175: 185-191)) are preferably used for directing expression of apolynucleotide preferentially in cells of the CNS. Preferably, thepromoter is tissue specific and is essentially not active outside thecentral nervous system, or the activity of the promoter is higher in thecentral nervous system than in other cells or tissues. For example, thepromoter may be specific for the spinal cord, brainstem, (medulla, pons,and midbrain), cerebellum, diencephalon (thalamus, hypothalamus),telencephalon (corpus stratium, cerebral cortex, or within the cortex,the occipital, temporal, parietal or frontal lobes), or combinations,thereof. The promoter may be specific for particular cell types, such asneurons or glial cells in the CNS. If it is active in glial cells, itmay be specific for astrocytes, oligodendrocytes, ependymal cells,Schwann cells, or microglia. If it is active in neurons, it may bespecific for particular types of neurons, e.g., motor neurons, sensoryneurons, or interneurons. The promoter may be specific for cells inparticular regions of the brain, for example, the cortex, stratium,nigra and hippocampus.

Suitable neuronal specific promoters include, but are not limited to,neuron specific enolase (NSE; Olivia et al (1991) Genomics 10: 157-165;GenBank Accession No: X51956), and human neurofilament light chainpromoter (NEFL; Rogaev et al (1992) Hum. Mol. Genet. 1: 781; GenBankAccession No: L04147). Glial specific promoters include, but are notlimited to, glial fibrillary acidic protein (GFAP) promoter (Morii et al(1991); GenBank Accession No: M65210), S100 promoter (Morii et al(1991); GenBank Accession No: M65210) and glutamine synthase promoter(Van den et al (1991) Biochem. Biophys. Acta. 2: 249-251; GenBankAccession No: X59834). In a preferred embodiment, the gene is flankedupstream (i.e., 5′) by the neuron specific enolase (NSE) promoter. Inanother preferred embodiment, the gene of interest is flanked upstream(i.e., 5′) by the elongation factor 1 alpha (EF) promoter. A hippocampusspecific promoter that might be used is the hippocampus specificglucocorticoid receptor (GR) gene promoter.

Alternatively, for treatment of cancer of the heart, Svensson et al(1999) describes the delivery of recombinant genes to cardiomyocytes byintramyocardial injection or intracoronary infusion of cardiotropicvectors, such as recombinant adeno-associated virus vectors, resultingin transgene expression in murine cardiomyocytes in vivo (Svensson et al(1999) “Efficient and stable transduction of cardiomyocytes afterintramyocardial injection or intracoronary perfusion with recombinantadeno-associated virus vectors.” Circulation. 99: 201-5). Melo et alreview gene and cell-based therapies for heart disease (Melo et al(2004) “Gene and cell-based therapies for heart disease.” FASEB J.18(6): 648-63). An alternative preferred route of administration is viaa catheter or stent. Stents represent an attractive alternative forlocalized gene delivery, as they provide a platform for prolonged geneelution and efficient transduction of opposed arterial walls. This genedelivery strategy has the potential to decrease the systemic spread ofthe viral vectors and hence a reduced host immune response. Bothsynthetic and naturally occurring stent coatings have shown potential toallow prolonged gene elution with no significant adverse reaction(Sharif et al (2004) “Current status of catheter- and stent-based genetherapy.” Cardiovasc Res. 64(2): 208-16).

It may also be desirable to be able to temporally regulate expression ofthe polynucleotide in the cell. Thus, it may be desirable thatexpression of the polynucleotide is directly or indirectly (see below)under the control of a promoter that may be regulated, for example bythe concentration of a small molecule that may be administered to thepatient when it is desired to activate or, more likely, repress(depending upon whether the small molecule effects activation orrepression of the said promoter) expression of the antibody from thepolynucleotide. This may be of particular benefit if the expressionconstruct is stable, i.e., capable of expressing the compound (in thepresence of any necessary regulatory molecules), in the cell for aperiod of at least one week, one, two, three, four, five, six, eightmonths or one or more years. Thus the polynucleotide may be operativelylinked to a regulatable promoter. Examples of regulatable promotersinclude those referred to in the following papers: Rivera et al (1999)Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orallybioavailable drug, using two separate adenovirus or adeno-associatedvirus (AAV) vectors, one encoding an inducible human growth hormone(hGH) target gene, and the other a bipartite rapamycin-regulatedtranscription factor); Magari et al (1997) J Clin Invest 100(11),2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22(review of adeno-associated viral vectors); Bohl et al (1998) Blood92(5), 1512-7 (control by doxycycline in adeno-associated vector);Abruzzese et al (1996) J Mol Med 74(7), 379-92 (review of inductionfactors, e.g. hormones, growth factors, cytokines, cytostatics,irradiation, heat shock and associated responsive elements).

It is preferred that the patient to be treated is a human patient. Forveterinary use, however, the compound is typically administered as asuitably acceptable formulation in accordance with normal veterinarypractice and the veterinary surgeon will determine the dosing regimenand route of administration which will be most appropriate for aparticular animal.

The cancer to be treated may be selected from ovarian cancer, breastcancer, renal cancer, gastrointestinal cancer, brain cancer, lungcancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, coloncancer, liver cancer, cervical cancer, prostate cancer, non-Hodgkinlymphoma, Hodgkin lymphoma, and nasal NK/T-cell lymphoma.

It is preferred, however, that the cancer to be treated is ovariancancer or breast cancer, most preferably ovarian cancer.

In an embodiment, the invention further comprises administering to thepatient at least one additional anticancer agent. The method maycomprise administering to the individual a pharmaceutical compositioncontaining the compound comprising or consisting of the OPCMLpolypeptide or fragment or variant thereof, or nucleic acid moleculewhich encodes the OPCML polypeptide or fragment or variant, and thefurther anticancer agent. However, it is appreciated that the compoundand the further anticancer agent may be administered separately, forinstance by separate routes of administration. Thus it is appreciatedthat the compound and the at least one further anticancer agent can beadministered sequentially or (substantially) simultaneously. They may beadministered within the same pharmaceutical formulation or medicament orthey may be formulated and administered separately.

In an embodiment of the medical uses, the medicament containing thecompound may also comprise the at least one further anticancer agent.

In another embodiment of the medical uses, the individual to be treatedmay be one who is administered at least one further anticancer agent. Itis appreciated that the individual may be administered the furtheranticancer agent at the same time as the medicament containing thecompound comprising or consisting of the OPCML polypeptide or fragmentor variant thereof, or nucleic acid molecule which encodes the OPCMLpolypeptide or fragment or variant, although the individual may havebeen (or will be) administered the further anticancer agent before (orafter) receiving the medicament containing the compound.

The further anticancer agent may be selected from alkylating agentsincluding nitrogen mustards such as mechlorethamine (HN₂),cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil;ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa;alkyl sulphonates such as busulphan; nitrosoureas such as carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin(streptozotocin); and triazenes such as decarbazine (DTIC;dimethyltriazenoimidazole-carboxamide); antimetabolites including folicacid analogues such as methotrexate (amethopterin); pyrimidine analoguessuch as fluorouracil (5-fluorouracil; 5-FU), floxuridine(fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); andpurine analogues and related inhibitors such as mercaptopurine(6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) andpentostatin (2′-deoxycoformycin); natural products including vincaalkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxinssuch as etoposide and teniposide; antibiotics such as dactinomycin(actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin,bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymessuch as L-asparaginase; and biological response modifiers such asinterferon alphenomes; miscellaneous agents including platinumcoordination complexes such as cisplatin (cis-DDP) and carboplatin;anthracenedione such as mitoxantrone and anthracycline; substituted ureasuch as hydroxyurea; methyl hydrazine derivative such as procarbazine(N-methylhydrazine, MlH); and adrenocortical suppressant such asmitotane (o,p′-DDD) and aminoglutethimide; taxol andanalogues/derivatives; cell cycle inhibitors; proteosome inhibitors suchas Bortezomib (Velcade®); signal transductase (e.g. tyrosine kinase)inhibitors such as Imatinib (Glivec®), COX-2 inhibitors, and hormoneagonists/antagonists such as flutamide and tamoxifen.

The clinically used anticancer agents are typically grouped by mechanismof action: Alkylating agents, Topoisomerase I inhibitors, TopoisomeraseII inhibitors, RNA/DNA antimetabolites, DNA antimetabolites andAntimitotic agents. The US NIH/National Cancer Institute website lists122 compounds(http://dtp.nci.nih.gov/docs/cancer/searches/standard_mechanism.html),all of which may be used in conjunction with the compound. They includeAlkylating agents including Asaley, AZQ, BCNU, Busulfan,carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil,chlorozotocin, cis-platinum, clomesone, cyanomorpholino-doxorubicin,cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone,melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard,PCNU, piperazine, piperazinedione, pipobroman, porfiromycin,spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa,triethylenemelamine, uracil nitrogen mustard, Yoshi-864; anitmitoticagents including allocolchicine, Halichondrin B, colchicine, colchicinederivative, dolastatin 10, maytansine, rhizoxin, taxol, taxolderivative, thiocolchicine, trityl cysteine, vinblastine sulphate,vincristine sulphate; Topoisomerase I Inhibitors including camptothecin,camptothecin, Na salt, aminocamptothecin, 20 camptothecin derivatives,morpholinodoxorubicin; Topoisomerase II Inhibitors includingdoxorubicin, amonafide, m-AMSA, anthrapyrazole derivative,pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin,mitoxantrone, menogaril, N,N-dibenzyl daunomycin, oxanthrazole,rubidazone, VM-26, VP-16; RNA/DNA antimetabolites including L-alanosine,5-azacytidine, 5-fluorouracil, acivicin, 3 aminopterin derivatives, anantifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar,ftorafur (pro-drug), 5,6-dihydro-5-azacytidine, methotrexate,methotrexate derivative, N-(phosphonoacetyl)-L-aspartate (PALA),pyrazofurin, trimetrexate; DNA antimetabolites including, 3-HP,2′-deoxy-5-fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate,ara-C, 5-aza-2′-deoxycytidine, beta-TGDR, cyclocytidine, guanazole,hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole,thioguanine and thiopurine.

It is, however, preferred that the at least one further anticancer agentis selected from cisplatin, carboplatin, 5-fluorouracil, paclitaxel,mitomycin C, doxorubicin, gemcitabine, tomudex, pemetrexed,methotrexate, irinotecan, oxaliplatin, or combinations thereof.

When the further anticancer agent or combination of agents has beenshown to be particularly effective for a specific tumour type, it may bepreferred that the compound is used in combination with that furtheranticancer agent(s) to treat that specific tumour type.

In one preferred embodiment, if the cancer is one in which HER2 isupregulated and/or in which HER2 mediated-signaling is upregulated, theadditional anticancer agent may be a HER2 inhibitor.

In another embodiment, if the cancer is one in which FGFR1 isupregulated and/or in which FGFR1 mediated-signaling is upregulated, theadditional anticancer agent is an FGFR1 inhibitor.

In yet another embodiment, if the cancer is one in which HER4 isupregulated and/or in which HER4 mediated-signaling is upregulated, theadditional anticancer agent is a HER4 inhibitor.

In a still further embodiment, if the cancer is one in which FGFR3 isupregulated and/or in which FGFR3 mediated-signaling is upregulated, theadditional anticancer agent is an FGFR3 inhibitor.

In a still yet further embodiment, if the cancer is one in which EPHA2is upregulated and/or in which EPHA2 mediated-signaling is upregulated,the additional anticancer agent is an EPHA2 inhibitor.

Suitable inhibitors of HER2, HER4, FGFR1, EPHA2 and FGFR3 includeantibodies that specifically or selectively bind to HER2, HER4, FGFR1,EPHA2 and FGFR3, respectively. Other inhibitors of HER2, HER4, FGFR1,EPHA2 and FGFR3 include siRNA, antisense polynucleotides and ribozymemolecules that are specific for polynucleotides encoding the respectiveHER2, HER4, FGFR1, EPHA2 and FGFR3 polypeptide, and which prevent theirexpression. Other inhibitors include small molecule inhibitors of thetyrosine kinase receptors, such as lapatinib, which typically act on theC-terminal kinase domain, inhibiting autophosphorylation and activationof the receptor.

It is appreciated that polynucleotide inhibitors of HER2, HER4, FGFR1,EPHA2 and FGFR3 may be administered directly, or may be administered inthe form of a polynucleotide that encodes the inhibitor. Thus, as usedherein, unless the context demands otherwise, by administering to theindividual an inhibitor of HER2, HER4, FGFR1, EPHA2 and FGFR3 which is apolynucleotide, we include the meanings of administering the inhibitordirectly, or administering a polynucleotide that encodes the inhibitor,typically in the form of a vector. Similarly, as used herein, unless thecontext demands otherwise, by a medicament or a composition comprisingan inhibitor of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 which is apolynucleotide, we include the meanings that the medicament orcomposition comprises the inhibitor itself, or comprises apolynucleotide that encodes the inhibitor.

Antibodies that specifically or selectively bind to HER2, HER4, FGFR1,EPHA2 or FGFR3 are also referred to as anti-HER2, anti-HER4, anti-FGFR1,anti-EPHA2 and anti-FGFR3 antibodies. Such antibodies will bind theirtarget with a greater affinity than for an irrelevant polypeptide, suchas human serum albumin (HSA). Preferably, the antibody binds the HER2,HER4, FGFR1, EPHA2 or FGFR3 with at least 5, or at least 10 or at least50 times greater affinity than for the irrelevant polypeptide. Morepreferably, the antibody molecule binds the HER2, HER4, FGFR1, EPHA2 orFGFR3 with at least 100, or at least 1,000, or at least 10,000 timesgreater affinity than for the irrelevant polypeptide. Such binding maybe determined by methods well known in the art, such as one of theBiacore® systems.

It is preferred if the antibodies have an affinity for HER2, HER4,FGFR1, EPHA2 or FGFR3 of at least 10⁻⁷ M and more preferably 10⁻⁸ M,although antibodies with higher affinities, e.g. 10⁻⁹ M, or higher, maybe even more preferred.

Typically, the antibody that selectively binds HER2, HER4, FGFR1, EPHA2or FGFR3 polypeptide binds to the extracellular region of thepolypeptide.

Preferably, when the antibody is administered to an individual, theantibody binds to the target HER2, HER4, FGFR1, EPHA2 or FGFR3 or to thespecified portion thereof with a greater affinity than for any othermolecule in the individual. Preferably, the antibody binds to (aspecified portion of) the HER2, HER4, FGFR1, EPHA2 or FGFR3 with atleast 2, or at least 5, or at least 10 or at least 50 times greateraffinity than for any other molecule in the individual. More preferably,the agent binds the HER2, HER4, FGFR1, EPHA2 or FGFR3 (at the specificdomain) with at least 100, or at least 1,000, or at least 10,000 timesgreater affinity than any other molecule in the individual. Preferably,the antibody molecule selectively binds the HER2, HER4, FGFR1, EPHA2 orFGFR3 without significantly binding other polypeptides in the body, suchas for example other RTKs, although in practice there may be some crossreactivity, especially between HER2 and HER4, and FGFR1, EPHA2 andFGFR3.

The term “antibody” or “antibody molecule” as used herein includes butis not limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments and fragments produced by a Fab expression library. Suchfragments include fragments of whole antibodies which retain theirbinding activity for a target substance, Fv, F(ab′) and F(ab′)2fragments, as well as single chain antibodies (scFv), fusion proteinsand other synthetic proteins which comprise the antigen-binding site ofthe antibody. The term also includes antibody-like molecules which maybe produced using phage-display techniques or other random selectiontechniques for molecules which bind to the specified polypeptide or toparticular regions of it. Thus, the term antibody includes all moleculeswhich contain a structure, preferably a peptide structure, which is partof the recognition site (i.e. the part of the antibody that binds orcombines with the epitope or antigen) of a natural antibody.Furthermore, the antibodies and fragments thereof may be humanisedantibodies, which are now well known in the art.

By antibodies we also include Nanobodies® (Ablynx) which areantibody-derived therapeutic proteins that contain the structural andfunctional properties of naturally-occurring heavy-chain antibodies. TheNanobody® technology was developed following the discovery thatcamelidae (camels and llamas) possess fully functional antibodies thatlack light chains. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (C_(H)2 and C_(H)3). Thecloned and isolated VHH domain is a perfectly stable polypeptideharbouring the full antigen-binding capacity of the original heavy-chainantibody. These VHH domains with their unique structural and functionalproperties form the basis of Nanobodies®. They combine the advantages ofconventional antibodies (high target specificity, high target affinityand low inherent toxicity) with important features of small moleculedrugs (the ability to inhibit enzymes and access receptor clefts).Furthermore, they are stable, have the potential to be administered bymeans other than injection, are easier to manufacture, and can behumanised. (See, for example U.S. Pat. No. 5,840,526; U.S. Pat. No.5,874,541; U.S. Pat. No. 6,005,079,U.S. Pat. No. 6,765,087; EP 1 589107; WO 97/34103; WO97/49805; U.S. Pat. No. 5,800,988; U.S. Pat. No.5,874,541 and U.S. Pat. No. 6,015,695).

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide. Engineeredantibodies, such as ScFv antibodies, can be made using the techniquesand approaches long known in the art. The advantages of using antibodyfragments, rather than whole antibodies, are several-fold. The smallersize of the fragments may lead to improved pharmacological properties,such as better penetration to the target site. Effector functions ofwhole antibodies, such as complement binding, are removed. Fab, Fv, ScFvand dAb antibody fragments can all be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of thefragments. Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By“bivalent” we mean that the antibodies and F(ab′)₂ fragments have twoantigen combining sites. In contrast, Fab, Fv, ScFv and dAb fragmentsare monovalent, having only one antigen combining site.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse,rabbit, goat, horse, etc) is immunised with an immunogenic polypeptidebearing a desired epitope(s), optionally haptenised to anotherpolypeptide. Depending on the host species, various adjuvants may beused to increase immunological response. Such adjuvants include, but arenot limited to, Freund's, mineral gels such as aluminium hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Serum from the immunised animal is collected and treatedaccording to known procedures. If serum containing polyclonal antibodiesto the desired epitope contains antibodies to other antigens, thepolyclonal antibodies can be purified by immunoaffinity chromatography.Techniques for producing and processing polyclonal antisera are wellknown in the art.

Monoclonal antibodies directed against entire polypeptides or particularepitopes thereof can also be readily produced by one skilled in the art.The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. Panels of monoclonal antibodies produced against the polypeptideslisted above can be screened for various properties; i.e., for isotypeand epitope affinity. Monoclonal antibodies may be prepared using any ofthe well known techniques which provides for the production of antibodymolecules by continuous cell lines in culture.

It is preferred if the antibody is a monoclonal antibody. In somecircumstance, particularly if the antibody is going to be administeredrepeatedly to a human patient, it is preferred if the monoclonalantibody is a human monoclonal antibody or a humanised monoclonalantibody, which are suitable for administration to humans withoutengendering an immune response by the human against the administeredimmunoglobulin. Suitably prepared non-human antibodies can be“humanised” in known ways, for example by inserting the CDR regions ofmouse antibodies into the framework of human antibodies. Humanisedantibodies can be made using the techniques and approaches described inVerhoeyen et al (1988) Science, 239, 1534-1536, and in Kettleborough etal, (1991) Protein Engineering, 14(7), 773-783. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. In general, the humanised antibodywill contain variable domains in which all or most of the CDR regionscorrespond to those of a non-human immunoglobulin, and framework regionswhich are substantially or completely those of a human immunoglobulinconsensus sequence.

Completely human antibodies may be produced using recombinanttechnologies. Typically large libraries comprising billions of differentantibodies are used. In contrast to the previous technologies employingchimerisation or humanisation of e.g. murine antibodies this technologydoes not rely on immunisation of animals to generate the specificantibody. Instead the recombinant libraries comprise a huge number ofpre-made antibody variants wherein it is likely that the library willhave at least one antibody specific for any antigen. Thus, using suchlibraries, an existing antibody having the desired bindingcharacteristics can be identified. In order to find the good binder in alibrary in an efficient manner, various systems where phenotype i.e. theantibody or antibody fragment is linked to its genotype i.e. theencoding gene have been devised. The most commonly used such system isthe so called phage display system where antibody fragments areexpressed, displayed, as fusions with phage coat proteins on the surfaceof filamentous phage particles, while simultaneously carrying thegenetic information encoding the displayed molecule (McCafferty et al,1990, Nature 348: 552-554). Phage displaying antibody fragments specificfor a particular antigen may be selected through binding to the antigenin question. Isolated phage may then be amplified and the gene encodingthe selected antibody variable domains may optionally be transferred toother antibody formats, such as e.g. full-length immunoglobulin, andexpressed in high amounts using appropriate vectors and host cells wellknown in the art. Alternatively, the “human” antibodies can be made byimmunising transgenic mice which contain, in essence, humanimmunoglobulin genes (Vaughan et al (1998) Nature Biotechnol. 16,535-539).

It is appreciated that when the antibody is for administration to anon-human individual, the antibody may have been specificallydesigned/produced for the intended recipient species.

The format of displayed antibody specificities on phage particles maydiffer. The most commonly used formats are Fab (Griffiths et al, 1994.EMBO J. 13: 3245-3260) and single chain (scFv) (Hoogenboom et al, 1992,J Mol Biol. 227: 381-388) both comprising the variable antigen bindingdomains of antibodies. The single chain format is composed of a variableheavy domain (V_(H)) linked to a variable light domain (V_(L)) via aflexible linker (U.S. Pat. No. 4,946,778). Before use as a therapeuticagent, the antibody may be transferred to a soluble format e.g. Fab orscFv and analysed as such. In later steps the antibody fragmentidentified to have desirable characteristics may be transferred into yetother formats such as full-length antibodies.

WO 98/32845 and Soderlind et al (2000) Nature BioTechnol. 18: 852-856describe technology for the generation of variability in antibodylibraries. Antibody fragments derived from this library all have thesame framework regions and only differ in their CDRs. Since theframework regions are of germline sequence the immunogenicity ofantibodies derived from the library, or similar libraries produced usingthe same technology, are expected to be particularly low (Soderlind etal, 2000). This property is of great value for therapeutic antibodies,reducing the risk that the patient forms antibodies to the administeredantibody, thereby reducing risks for allergic reactions, the occurrenceof blocking antibodies, and allowing a long plasma half-life of theantibody. Thus, when developing therapeutic antibodies to be used inhumans, modern recombinant library technology (Soderlind et al., 2001,Comb. Chem. & High Throughput Screen. 4: 409-416) is now used inpreference to the earlier hybridoma technology.

In another embodiment, the HER2, HER4, FGFR1, EPHA2 or FGFR3 inhibitormay be a small interfering RNA (siRNA) (described by Hannon et al.Nature, 418 (6894): 244-51 (2002); Brummelkamp et al., Science 21, 21(2002); and Sui et al., Proc. Natl. Acad. Sci. USA 99, 5515-5520(2002)). RNA interference (RNAi) is the process of sequence-specificpost-transcriptional gene silencing in animals initiated bydouble-stranded (dsRNA) that is homologous in sequence to the silencedgene. The mediators of sequence-specific mRNA degradation are typically21- and 22-nucleotide small interfering RNAs (siRNAs) which, in vivo,may be generated by ribonuclease III cleavage from longer dsRNAs.21-nucleotide siRNA duplexes have been shown to specifically suppressexpression of both endogenous and heterologous genes (Elbashir et al(2001) Nature 411: 494-498). In mammalian cells it is considered thatthe siRNA has to be comprised of two complementary 21 mers as describedbelow since longer double-stranded (ds) RNAs will activate PKR(dsRNA-dependent protein kinase) and inhibit overall protein synthesis.

Duplex siRNA molecules selective for a polynucleotide encoding the HER2,HER4, FGFR1, EPHA2 or FGFR3 polypeptide can readily be designed byreference to its cDNA sequence. For example, they can be designed byreference to the HER2, HER4, FGFR1, EPHA2 or FGFR3 cDNA sequences in theGenbank Accession Nos listed above.

Typically, the first 21-mer sequence that begins with an AA dinucleotidewhich is at least 120 nucleotides downstream from the initiatormethionine codon is selected. The RNA sequence perfectly complementaryto this becomes the first RNA oligonucleotide. The second RNA sequenceshould be perfectly complementary to the first 19 residues of the first,with an additional UU dinucleotide at its 3′ end. Once designed, thesynthetic RNA molecules can be synthesized using methods well known inthe art.

A number of suitable anti-HER2, HER4, FGFR1, EPHA2 and FGFR3 siRNAs arecommercially available, for example, from Sigma-Aldrich in the form ofMISSION® Lentiviral transduction particles.

siRNAs may be introduced into cells in the patient using any suitablemethod, such as those described herein. Typically, the RNA is protectedfrom the extracellular environment, for example by being containedwithin a suitable carrier or vehicle. Liposome-mediated transfer, e.g.the oligofectamine method, may be used.

In another embodiment, the HER2, HER4, FGFR1, EPHA2 or FGFR3 inhibitormay be an antisense polynucleotide.

Antisense nucleic acid molecules selective for a polynucleotide encodingthe HER2, HER4, FGFR1, EPHA2 or FGFR3 polypeptide can readily bedesigned by reference to its cDNA or gene sequence, as is known in theart. Antisense nucleic acids, such as oligonucleotides, aresingle-stranded nucleic acids, which can specifically bind to acomplementary nucleic acid sequence. By binding to the appropriatetarget sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed.These nucleic acids are often termed “antisense” because they arecomplementary to the sense or coding strand of the gene. Recently,formation of a triple helix has proven possible where theoligonucleotide is bound to a DNA duplex. It was found thatoligonucleotides could recognize sequences in the major groove of theDNA double helix. A triple helix was formed thereby. This suggests thatit is possible to synthesize a sequence-specific molecule whichspecifically binds double-stranded DNA via recognition of major groovehydrogen binding sites. By binding to the target nucleic acid, the aboveoligonucleotides can inhibit the function of the target nucleic acid.This could, for example, be a result of blocking the transcription,processing, poly(A) addition, replication, translation, or promotinginhibitory mechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and thenintroduced into cells, for example by microinjection or uptake from thecell culture medium into the cells, or they are expressed in cells aftertransfection with plasmids or retroviruses or other vectors carrying anantisense gene. Antisense oligonucleotides were first discovered toinhibit viral replication or expression in cell culture for Rous sarcomavirus, vesicular stomatitis virus, herpes simplex virus type 1, simianvirus and influenza virus. Since then, inhibition of mRNA translation byantisense oligonucleotides has been studied extensively in cell-freesystems including rabbit reticulocyte lysates and wheat germ extracts.Inhibition of viral function by antisense oligonucleotides has beendemonstrated ex vivo using oligonucleotides which were complementary tothe AIDS HIV retrovirus RNA (Goodchild (1988) “Inhibition of HumanImmunodeficiency Virus Replication by Antisense Oligodeoxynucleotides”,Proc. Natl. Acad. Sci. (USA) 85(15): 5507-11). The Goodchild studyshowed that oligonucleotides that were most effective were complementaryto the poly(A) signal; also effective were those targeted at the 5′ endof the RNA, particularly the cap and 5N untranslated region, next to theprimer binding site and at the primer binding site. The cap, 5′untranslated region, and poly(A) signal lie within the sequence repeatedat the ends of retrovirus RNA (R region) and the oligonucleotidescomplementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. Forexample, 20-mer oligonucleotides have been shown to inhibit theexpression of the epidermal growth factor receptor mRNA (Witters et al.,Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotideshave been shown to decrease the expression of adrenocorticotropichormone by greater than 90% (Frankel et al., J Neurosurg 91:261-7(1999)). However, it is appreciated that it may be desirable to useoligonucleotides with lengths outside this range, for example 10, 11,12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

Antisense polynucleotides may be administered systemically.Alternatively, and preferably, the inherent binding specificity ofpolynucleotides characteristic of base pairing is enhanced by limitingthe availability of the polynucleotide to its intended locus in vivo,permitting lower dosages to be used and minimizing systemic effects.Thus, polynucleotides may be applied locally to the solid tumor toachieve the desired effect. The concentration of the polynucleotides atthe desired locus is much higher than if the polynucleotides wereadministered systemically, and the therapeutic effect can be achievedusing a significantly lower total amount. The local high concentrationof polynucleotides enhances penetration of the targeted cells andeffectively blocks translation of the target nucleic acid sequences.

It will be appreciated that antisense agents may also include largermolecules which bind to polynucleotides (mRNA or genes) encoding theHER2, HER4, FGFR1 or FGFR3 polypeptide and substantially preventexpression of the protein. Thus, antisense molecules which aresubstantially complementary to the respective mRNA are also envisaged.

The molecules may be expressed from any suitable genetic construct anddelivered to the patient. Typically, the genetic construct whichexpresses the antisense molecule comprises at least a portion of theHER2, HER4, FGFR1, EPHA2 or FGFR3 cDNA or gene operatively linked to apromoter which can express the antisense molecule in the cell.Preferably, the genetic construct is adapted for delivery to a humancell.

In another embodiment, the HER2, HER4, FGFR1, EPHA2 or FGFR3 inhibitormay be a ribozyme. Ribozymes are RNA or RNA-protein complexes thatcleave nucleic acids in a site-specific fashion. Ribozymes have specificcatalytic domains that possess endonuclease activity. For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate. This specificity has beenattributed to the requirement that the substrate binds via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids.For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes canact as endonucleases with a sequence specificity greater than that ofknown ribonucleases and approaching that of the DNA restriction enzymes.Thus, sequence-specific ribozyme-mediated inhibition of gene expressionmay be particularly suited to therapeutic applications, and ribozymesspecific for a polynucleotide encoding the HER2, HER4, FGFR1, EPHA2 orFGFR3 polypeptide may be designed by reference to the HER2, HER4, FGFR1,EPHA2 or FGFR3 cDNA sequence, respectively.

Methods and routes of administering polynucleotide inhibitors, such assiRNA molecules, antisense molecules and ribozymes, to a patient, aredescribed in more detail below.

Typically, the HER2 inhibitor is an anti-HER2 antibody. Preferredanti-HER2 antibodies include trastuzumab and pertuzumab.

Trastuzumab (Herceptin®) is presented as a white to pale yellowlyophilized powder concentrate for solution for infusion. It iscurrently approved for the treatment of patients with metastatic breastcancer whose tumors overexpress HER2: (a) as monotherapy for thetreatment of those patients who have received at least two chemotherapyregimens for their metastatic disease in which the prior chemotherapyhas included at least an anthracycline and a taxane unless patients areunsuitable for these treatments, and hormone receptor positive patientshave failed hormonal therapy unless patients are unsuitable for thesetreatments; and (b) in combination with paclitaxel for the treatment ofthose patients who have not received chemotherapy for their metastaticdisease and for whom an anthracycline is not suitable. Trastuzumab ispreferably used in patients whose tumors have HER2 overexpression at a3+ level as determined by immunohistochemistry.

The recommended dosage scheme consists of trastuzumab loading (4 mg/kgbody weight) and subsequent weekly doses of 2 mg/kg body weight. It istypically administered until progression of the disease.

Other preferred HER2 inhibitors include lapatinib, neratinib and BIBW2992.

Lapatinib (Tykerb®) is a small molecule and a member of the4-anilinoquinazoline class of kinase inhibitors. It is present as themonohydrate of the ditosylate salt, with chemical name N-(3chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)-6-[5-({[2(methylsulfonyl)ethyl]amino}methyl)-2-furanyl]-4-quinazolinaminebis (4 methylbenzenesulfonate) monohydrate. It has the molecular formulaC₂₉H₂₆ClFN₄O₄S(C₇H₈O₃S)₂H₂O and a molecular weight of 943.5. Lapatinibis a yellow solid, and its solubility in water is 0.007 mg/ml and in0.1N HCl is 0.001 mg/mL at 25° C. Each 250 mg tablet of Tykerb® contains405 mg of lapatinib ditosylate monohydrate, equivalent to 398 mg oflapatinib ditosylate or 250 mg lapatinib free base. The recommended doseof Tykerb® is 1,250 mg (5 tablets) given orally once daily on Days 1-21continuously, usually in combination with capecitabine 2,000 mg/m²/day(administered orally in 2 doses approximately 12 hours apart) on Days1-14 in a repeating 21 day cycle. Lapatinib is preferably taken at leastone hour before or one hour after a meal. The dose of Lapatinib ispreferably taken once daily; dividing the daily dose is not recommended

Preferably, the FGFR1 inhibitor is selected from the group consisting ofPD173074, SU5402 and Indirubin-3′-monoxime.

Such agents can be used in combination with the compounds describedabove to sensitize cancers with upregulated FGFR1, or with upregulatedFGFR1-signalling, thus providing additional efficacy. —Further suitableagents include AKT inhibitors, DNA damaging agents such as platinum, andalso radiotherapy.

The HER4 inhibitor may be lapatinib, which is also a HER2 inhibitor;PF-00299804 (Pfizer) which is a pan-HER inhibitor that inhibits HER1,HER2 and HER4; AEE-788 (Novartis), which is also a HER2 inhibitor; andCI-1033 (Pfizer), which is a pan-HER and an EGFR inhibitor.

The FGFR3 inhibitor may be PKC 412; PD173074, which is also an FGFR1inhibitor; or an anti-FGFR3 monoclonal antibody such as those describedby Qing et al in which R3Mab was the most effective antagonist of FGFR3activity and was shown to have antitumor activity (Qing et al (2009)“Antibody-based targeting of FGFR3 in bladder carcinoma andt(4;14)-positive multiple myeloma in mice” J. Clin. Invest. 119(5):1216-1229).

The EPHA2 inhibitor may be a soluble EphA2 receptor, which has beenshown to inhibit tumor angiogenesis and progression in vivo (Brantley etal (2002) “Soluble Eph A receptors inhibit tumor angiogenesis andprogression in vivo.” Oncogene 21(46): 7011-26. The EPHA2 inhibitor mayalso be a catechol derivative described by Stroylov et al (2010) “Novelfragment-like inhibitors of EphA2 obtained by experimental screening andmodeling” Mendeleev Communications 20(5): 263-265.

We have observed that administration of recombinant human OPCML tocancer cells inhibits phospho S473 AKT, suggesting a potential synergywith platinum based chemotherapy and ionizing radiation.

Thus, in one preferred embodiment, the further anticancer agent may be aplatinum-based chemotherapeutic agent. Clinically approvedplatinum-based chemotherapeutic agents include carboplatin(cis-diammine(cyclobutane-1,1-dicarboxylate-O,O′)platinum(II); CASRegistry Number 41575-94-4), cisplatin((SP-4-2)-diamminedichloridoplatinum; CAS Registry number 15663-271),and oxaliplatin([(1R,2R)-cyclohexane-1,2-diamine]ethanedioato-O,O′)platinum(II); CASRegistry Number 63121-00-6). Oxaliplatin may also be typicallyadministered with fluorouracil and leucovorin in a combination known asFOLFOX. These platinum-based chemotherapeutic agents are typicallyadministered intravenously as a short-term infusion in physiologicalsaline, as is well known in the art.

Additionally or alternatively, the method may further compriseadministering an AKT inhibitor to the patient. Many suitable AKTinhibitors are known in the art and include A-443654 by AbbottLaboratories (Han et al (2007) Oncogene 26: 5655-5661); GSK690693 byGlaxoSmithKline (Levy et al (2009) Blood 113(8): 1723-29); GSK2110183;GSK2141795; triciribine (Merck); KP372-1 (Mandal et al (2006) OralOncol. 42(4): 430-439); VQD-002 by VioQuest Pharmaceuticals; andperifosine (Gills et al (2009) Current Oncology Reports 11: 102-110).

In another embodiment, the patient may be one who is being, or who hasbeen, treated with radiotherapy.

We have shown that the Ig2 domain of OPCML binds to and interacts withFGFR1. Thus, in a specific embodiment of this aspect of the invention,the cancer is one in which FGFR1 is upregulated and/or in which FGFR1mediated-signaling is upregulated, and the fragment of the OPCMLpolypeptide comprises or consists of the Ig2 domain of OPCML (SEQ ID NO:2).

Thus this aspect of the invention includes a method of treating apatient having a cancer in which FGFR1 is upregulated and/or in whichFGFR1 mediated-signaling is upregulated, the method comprisingadministering to the patient a compound comprising or consisting of apolypeptide comprising the Ig2 domain of OPCML (SEQ ID NO: 2), or avariant thereof having at least 90% sequence identity with SEQ ID NO: 2,or a nucleic acid molecule which encodes the polypeptide or variantthereof.

This aspect of the invention also provides the use of a compoundcomprising or consisting of a polypeptide comprising the Ig2 domain ofOPCML (SEQ ID NO: 2), or a variant thereof having at least 90% sequenceidentity with SEQ ID NO: 2, or a nucleic acid molecule which encodes thepolypeptide or variant thereof, in the preparation of a medicament fortreating a patient having a cancer in which FGFR1 is upregulated and/orin which FGFR1 mediated-signaling is upregulated.

This aspect of the invention also provides a compound comprising orconsisting of a polypeptide comprising the Ig2 domain of OPCML (SEQ IDNO: 2), or a variant thereof having at least 90% sequence identity withSEQ ID NO: 2, or a nucleic acid molecule which encodes the polypeptideor variant thereof, for use in treating a patient having a cancer inwhich FGFR1 is upregulated and/or in which FGFR1 mediated-signaling isupregulated.

It is, therefore, appreciated that the invention includes apharmaceutical composition comprising a fragment of OPCML that comprisesor consists of the Ig2 domain of OPCML (residues 136-219 of humanOPCML); such a fragment of OPCML for use as a medicament; the use ofsuch a fragment of OPCML in the preparation of a medicament for treatingcancer; a method of treating cancer by administering such a fragment ofOPCML to a patient; and such a fragment of OPCML for use in treatingcancer.

We have also shown that the Ig3 domain of OPCML binds to and interactswith HER2. Thus, in another specific embodiment of this aspect of theinvention, the cancer is one in which HER2 is upregulated and/or inwhich HER2 mediated-signaling is upregulated, and the fragment of theOPCML polypeptide comprises or consists of the Ig3 domain of OPCML (SEQID NO: 3).

Thus this aspect of the invention includes a method of treating apatient having a cancer in which HER2 is upregulated and/or in whichHER2 mediated-signaling is upregulated, the method comprisingadministering to the patient a compound comprising or consisting of apolypeptide comprising the Ig3 domain of OPCML (SEQ ID NO: 3), or avariant thereof having at least 90% sequence identity with SEQ ID NO: 3,or a nucleic acid molecule which encodes the polypeptide or variantthereof.

This aspect of the invention also provides the use of a compoundcomprising or consisting of a polypeptide comprising the Ig3 domain ofOPCML (SEQ ID NO: 3), or a variant thereof having at least 90% sequenceidentity with SEQ ID NO: 3, or a nucleic acid molecule which encodes thepolypeptide or variant thereof, in the preparation of a medicament fortreating a patient having a cancer in which HER2 is upregulated and/orin which HER2 mediated-signaling is upregulated.

This aspect of the invention also provides a compound comprising orconsisting of a polypeptide comprising the Ig3 domain of OPCML (SEQ IDNO: 3), or a variant thereof having at least 90% sequence identity withSEQ ID NO: 3, or a nucleic acid molecule which encodes the polypeptideor variant thereof, for use in treating a patient having a cancer inwhich HER2 is upregulated and/or in which HER2 mediated-signaling isupregulated.

It is, therefore, appreciated that the invention includes apharmaceutical composition comprising a fragment of OPCML that comprisesor consists of the Ig3 domain of OPCML (residues 223-310 of humanOPCML); such a fragment of OPCML for use as a medicament; the use ofsuch a fragment of OPCML in the preparation of a medicament for treatingcancer; a method of treating cancer by administering such a fragment ofOPCML to a patient; and such a fragment of OPCML for use in treatingcancer.

The Ig1 domain of OPCML is required for dimerization of OPCML. Thus,optionally, especially if dimerization is required, the fragment of theOPCML polypeptide further comprises the Ig1 domain of OPCML (SEQ ID NO:4). Thus the compound may comprise a fusion of the Ig1 and Ig2 domains,or a fusion of the Ig1 and Ig3 domains of OPCML.

Alternatively, the fragment of the OPCML polypeptide may be fused to anantibody Fc fragment. This would also allow dimerization if required.Thus the compound may comprise an Ig1-Fc fusion of an Ig2-Fc fusion, oran Ig1-Fc fusion of an Ig3-Fc fusion.

It is appreciated that a compound comprising or consisting of the Ig2domain, but not the Ig3 domain of OPCML, can be used to target FGFR1whilst not targeting HER2. Conversely, a compound comprising orconsisting of the Ig3 domain, but not the Ig2 domain of OPCML, can beused to target HER2 but not FGFR1.

In a related embodiment, the invention provides a method of treating apatient having HER2-positive (HER2+) cancer, the method comprisingadministering to the patient a compound comprising or consisting of anOPCML polypeptide (SEQ ID NO: 1), or a fragment thereof which comprisesthe Ig3 domain of OPCML (SEQ ID NO: 3), or a variant thereof having atleast 90% sequence identity with the OPCML polypeptide or the fragmentthereof, or a nucleic acid molecule which encodes the OPCML polypeptideor fragment or variant thereof; and a HER2 inhibitor.

This embodiment of the invention also provides a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises the Ig3 domain of OPCML (SEQ ID NO: 3), or a variantthereof having at least 90% sequence identity with the OPCML polypeptideor the fragment thereof, or a nucleic acid molecule which encodes theOPCML polypeptide or fragment or variant thereof, and a HER2 inhibitor,for use in treating a patient having HER2-positive (HER2+) cancer.

In an embodiment, for the combination for use in treating a patienthaving HER2+ cancer, the compound and the HER2 inhibitor may beadministered separately, whether temporally separated, or administeredsubstantially simultaneously.

In an alternative embodiment, for the combination for use in treating apatient having HER2+ cancer, the compound and the HER2 inhibitor may beadministered within the same formulation.

The invention further provides the use of a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises the Ig3 domain of OPCML (SEQ ID NO: 3), or a variantthereof having at least 90% sequence identity with the OPCML polypeptideor the fragment thereof, or a nucleic acid molecule which encodes theOPCML polypeptide or fragment or variant thereof, and a HER2 inhibitor,in the preparation of a medicament treating a patient havingHER2-positive (HER2+) cancer.

In an embodiment, the variant of the fragment of OPCML which comprisesthe Ig3 domain of OPCML has at least 91% sequence identity, or at least92% sequence identity, or at least 93% sequence identity, or at least94% sequence identity, or at least 95% sequence identity, or at least96% sequence identity, or at least 97% sequence identity, or at least98% sequence identity, or at least 99% sequence identity, with theequivalent region of the human OPCML polypeptide fragment. Suchfragments and variants, and nucleic acid molecules encoding them, may bemade, for example, using the methods of recombinant DNA technology,protein engineering and site-directed mutagenesis, which are well knownin the art.

It is preferred that the fragment of OPCML which comprises the Ig3domain of OPCML, or the variant thereof possesses at least 50% of theactivity of full length human OPCML in inhibiting the proliferation ofHER2+ cancer cells in vitro. It is more preferred if the fragment ofOPCML, or the variant of the OPCML or fragment thereof possesses atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100% or more of the activity of full length humanOPCML in inhibiting the proliferation of HER2+ cancer cells in vitro.This can be determined using methods well known in the art and describedin the Examples below.

As discussed above, it is appreciated that the fragment of the OPCMLpolypeptide or variant thereof further comprises the Ig1 domain of OPCML(SEQ ID NO: 4) or may be fused to an antibody Fc fragment.

Preferences for the formulation and routes of administration of thecompound are as described above.

Preferences for the HER2 inhibitor are as defined above, with anti-HER2antibody such as trastuzumab or pertuzumab, or lapatinib, neratinib andBIBW 2992, being most preferred.

By a HER2+ cancer we mean a cancer that is HercepTest™ positive or FISHpositive.

HER2 immunohistochemistry (IHC) may be performed using the DakoHercepTest™ kit, which is a semi-quantitative immunohistochemical assayfor determination of HER2 protein (c-erbB-2 oncoprotein) overexpressionin breast cancer tissues routinely processed for histologicalevaluation. HercepTest™ is intended as an aid in the assessment ofpatients for whom treatment with humanized monoclonal antibody to HER2protein, Herceptin™ (trastuzumab), is being considered. HercepTest™specifically demonstrates overexpression of HER2 protein. Positive ornegative results aid in the classification of abnormal cells/tissues andprovide a basis for treatment selection. The kit includes reagentsrequired for the immunohistochemical staining (except wash buffer),control slides representing different expression levels of HER2 protein,and detailed instructions. A result of 3+ in using the Dako HercepTest™kit is indicative of a HER+ cancer.

A HER2+ positive cancer is one in which the HER2 gene copy number isfound to be greater than two by FISH analysis.

The HER2+ cancer may be ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, colon cancer, liver cancer,cervical cancer, prostate cancer, non-Hodgkin lymphoma, Hodgkinlymphoma, and nasal NK/T-cell lymphoma.

Most preferably, the cancer is ovarian cancer or breast cancer orgastric cancer.

Typically, the compound comprising or consisting of the OPCML orfragment or variant or nucleic acid will be administered separately fromthe HER2 inhibitor. Typically, the compound will be administered givenas a separate infusion, either substantially simultaneously with, orbefore or after the inhibitor. It is possible that when both theinhibitor, e.g., trastuzumab, and the compound are to be administered inthe same way, e.g. by infusion, they may be administered together.Lapatinib is a tablet so will usually be administered separately fromthe compound.

A second aspect of the invention provides a method of treating a patienthaving OPCML-positive (OPCML+) and HER2 positive (HER2+) cancer, themethod comprising administering a HER2 inhibitor to the patient.

This aspect of the invention provides the use of a HER2 inhibitor in thepreparation of a medicament for treating a patient having OPCML+ andHER2+ cancer. This aspect of the invention also provides a HER2inhibitor for use in treating a patient having OPCML+ and HER2+ cancer.

Preferences for the HER2 inhibitor are as defined above, with anti-HER2antibody such as trastuzumab or pertuzumab, or lapatinib, neratinib andBIBW 2992, being most preferred.

In an embodiment, the invention further comprises the prior step ofidentifying a patient having OPCML+ and HER2+ cancer. In an alternativeembodiment, the invention further comprises the prior step ofdetermining whether the patient has OPCML+ and HER2+ cancer

The OPCML+ cancer may be identified by IHC. A cancer that shows 2+ or 3+positive cytoplasmic expression of OPCML by IHC in comparison tonon-cancerous adjacent tissue may be considered to be OPCML+. In anembodiment, by quantitative RT-PCR, OPCML+ cancer is one that has atleast 2×, or at least 3×, or at least 4×, or at least 5×, or at least10×, or at least 50×, or at least 100×, or at least 1000× above controllevels. Typically, the OPCML+ cancer has above-median, and preferablyupper-quartile, expression of OPCML.

In an embodiment, the control levels for OPCML in the cancer of aspecific tissue or organ may be the average (mean, or preferably median)level of OPCML found in the same tissue or organ in a population ofindividuals who do not have cancer in that tissue or organ, andtypically do not have cancer at all.

In an alternative embodiment, the control levels for OPCML in the cancerof a specific tissue or organ may be the average (mean, or preferablymedian) level of OPCML found in a population of patients with the samecancer.

Typically, the population of control individuals is matched for gender,age, and ethnic origin. Preferably, the population of controlindividuals comprises at least 5, 10, 50, 100, 200, 300, 400 or 500individuals and more preferably at least 1000, 5000 or 10000individuals.

Suitable methods for determining whether a cancer is HER2+ are known inthe art and are discussed above. For example, a HER2+ positive cancer isone in which the HER2 gene copy number is found to be greater than twoby FISH analysis.

The OPCML+ and HER2+ cancer may be ovarian cancer, breast cancer, renalcancer, gastrointestinal cancer, brain cancer, lung cancer,nasopharyngeal cancer, esophageal cancer, gastric cancer, colon cancer,liver cancer, cervical cancer, prostate cancer, non-Hodgkin lymphoma,Hodgkin lymphoma, and nasal NK/T-cell lymphoma.

Most preferably, the cancer is ovarian cancer or breast cancer.

In an embodiment, the method further comprises administering aplatinum-based chemotherapeutic agent to the patient. Preferences forthe platinum-based chemotherapeutic agents are as described above.Carboplatin, oxaliplatin and cisplatin are most preferred.

We have found that AKT s473 phosphorylation is a key event associatedwith platinum resistance, and targeting this reverses platinumresistance by altering AKT mediated cell survival. This has beendemonstrated in the context of using an AKT inhibitor. We have alsofound that administration of recombinant human OPCML inhibits AKT s473phosphorylation which suggests that OPCML reverses platinum resistancein this in vitro system. Thus, a third aspect of the invention providesa method of treating a patient having OPCML-positive (OPCML+) cancer,the method comprising administering a platinum-based chemotherapeuticagent to the patient.

This aspect of the invention provides the use of a platinum-basedchemotherapeutic agent in the preparation of a medicament for treating apatient having OPCML+ cancer. This aspect of the invention also providesa platinum-based chemotherapeutic agent for use in treating a patienthaving OPCML+ cancer.

Preferences for the platinum-based chemotherapeutic agents are asdescribed above. Carboplatin, oxaliplatin and cisplatin are mostpreferred.

In an embodiment, the invention further comprises the prior step ofidentifying a patient having OPCML+ cancer. In an alternativeembodiment, the invention further comprises the prior step ofdetermining whether the patient has OPCML+ cancer. Suitable methods fordetermining whether a cancer is OPCML+ are known in the art and arediscussed above.

The OPCML+ cancer may be ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, colon cancer, liver cancer,cervical cancer, prostate cancer, non-Hodgkin lymphoma, Hodgkinlymphoma, and nasal NK/T-cell lymphoma.

Most preferably, the cancer is ovarian cancer or breast cancer.

A fourth aspect of the invention provides a method of treating a patienthaving OPCML-negative cancer, the method comprising administering

-   -   a compound comprising or consisting of an OPCML polypeptide (SEQ        ID NO: 1), or a fragment thereof which comprises at least one Ig        domain of OPCML, or a variant thereof having at least 90%        sequence identity with the OPCML polypeptide or the fragment        thereof, or a nucleic acid molecule which encodes the OPCML        polypeptide or fragment or variant thereof, and    -   a platinum-based chemotherapeutic agent, or ionizing radiation,        or both, to the patient.

This aspect of the invention includes the use of the compound comprisingor consisting of the OPCML polypeptide or fragment or variant thereof,or the nucleic acid molecule which encodes the OPCML polypeptide orfragment or variant, and a platinum-based chemotherapeutic agent, in thepreparation of a medicament for treating a patient having OPCML-negativecancer. The patient may be one who is being administered ionizingradiation (i.e., radiotherapy).

This aspect of the invention also includes a compound comprising orconsisting of the OPCML polypeptide or fragment thereof or variantthereof, or the nucleic acid molecule which encodes the OPCMLpolypeptide or fragment or variant, and a platinum-basedchemotherapeutic agent, for use in treating a patient havingOPCML-negative cancer. The patient may be one who is undergoingradiotherapy.

The OPCML-negative cancer may be identified by IHC. A cancer that shows0 or 1+ positive cytoplasmic expression of OPCML by IHC in comparison tonon-cancerous adjacent tissue may be considered to be OPCML-negative.

In an embodiment, by quantitative RT-PCR, OPCML-negative cancer is onethat has at least below control levels, e.g., below 50%, or below 25%,or below 10%, or below 5%, or below 1%, or below 0.1%, of OPCML controllevels for cancer of that specific tissue or organ as discussed above.Typically, the OPCML-negative cancer has below-median, and preferablylowest-quartile, expression of OPCML.

In an embodiment, in an OPCML-negative cancer, OPCML is not expressed orits expression is not at a detectable level.

Preferences for the compound comprising or consisting of the OPCMLpolypeptide or fragment thereof or variant thereof or the nucleic acidmolecule which encodes the OPCML polypeptide or fragment or variant, forthe platinum-based chemotherapeutic agents, and for formulations androutes of administration etc, are as described above in the first aspectof the invention.

In an embodiment, the invention further comprises the prior step ofidentifying a patient having OPCML-negative cancer. In an alternativeembodiment, the invention further comprises the prior step ofdetermining whether the patient has OPCML-negative cancer. Suitablemethods for determining whether a cancer is OPCML-negative are known inthe art and are discussed herein.

The OPCML-negative cancer may be ovarian cancer, breast cancer, renalcancer, gastrointestinal cancer, brain cancer, lung cancer,nasopharyngeal cancer, esophageal cancer, gastric cancer, colon cancer,liver cancer, cervical cancer, prostate cancer, non-Hodgkin lymphoma,Hodgkin lymphoma, and nasal NK/T-cell lymphoma.

Most preferably, the cancer is ovarian cancer or breast cancer.

A fifth aspect of the invention provides a method for selecting atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer in which HER2, HER4,        FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which HER2,        HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is        upregulated; and    -   selecting a treatment regime that includes administering to the        patient a compound comprising or consisting of an OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof which        comprises at least one Ig domain of OPCML, or a variant thereof        having at least 90% sequence identity with the OPCML polypeptide        or the fragment thereof, or a nucleic acid molecule which        encodes the OPCML polypeptide or fragment or variant thereof.

This aspect of the invention provides a method for selecting a treatmentregime for a cancer patient, the method comprising:

-   -   determining whether the patient has a cancer in which HER2,        HER4, FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which        HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is        upregulated; and    -   selecting a treatment regime that includes administering to the        patient a compound comprising or consisting of an OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof which        comprises at least one Ig domain of OPCML, or a variant thereof        having at least 90% sequence identity with the OPCML polypeptide        or the fragment thereof, or a nucleic acid molecule which        encodes the OPCML polypeptide or fragment or variant thereof.

This aspect of the invention provides a method for identifying atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer in which HER2, HER4,        FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which HER2,        HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is        upregulated; or    -   determining whether the patient has a cancer in which HER2,        HER4, FGFR1, EPHA2 and/or FGFR3 is upregulated and/or in which        HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mediated-signaling is        upregulated,    -   wherein an upregulated level of HER2, HER4, FGFR1, EPHA2 and/or        FGFR3, and/or an upregulated level of HER2, HER4, FGFR1, EPHA2        and/or FGFR3 mediated-signaling, indicates that a treatment        regime that includes administering a compound comprising or        consisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment        thereof which comprises at least one Ig domain of OPCML, or a        variant thereof having at least 90% sequence identity with the        OPCML polypeptide or the fragment thereof, or a nucleic acid        molecule which encodes the OPCML polypeptide or fragment or        variant thereof, will be more effective than a treatment regime        that does not include administering the OPCML polypeptide or        fragment or variant or nucleic acid molecule.

The upregulation of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 can bedetermined by assessing HER2, HER4, FGFR1, EPHA2 and/or FGFR3 mRNAlevels, polypeptide levels or gene copy number as described above and asis well known in the art.

Typically, the mRNA levels, polypeptide levels or gene copy number areassessed in a biopsy sample of the cancer. Alternatively, the mRNAlevels or polypeptide levels may be assessed in ascites malignant cellsfrom the patient.

For example, the level of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 that isindicative of upregulation may be a level of HER2, HER4, FGFR1, EPHA2and/or FGFR3 in the tissue or organ with the cancer that is equal to orgreater than the mean+2 standard deviations (SD) of the level of HER2,HER4, FGFR1, EPHA2 and/or FGFR3 found in the same tissue or organ in apopulation of control individuals who do not have cancer in that tissueor organ.

Preferably, however, the level of HER2, HER4, FGFR1, EPHA2 and/or FGFR3that is indicative of upregulation is an above-median level, or upperquartile level, of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 in the tissueor organ with the cancer in comparison to a population of individualshaving cancer in the tissue or organ.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forformulations and routes of administration, are as described above in thefirst aspect of the invention.

Thus, for example, if the cancer is one in which FGFR1 is upregulatedand/or in which FGFR1 mediated-signaling is upregulated, the fragment ofthe OPCML polypeptide comprises or consists of the Ig2 domain of OPCML(SEQ ID NO: 2), as discussed above.

In another embodiment, if the cancer is one in which HER2 is upregulatedand/or in which HER2 mediated-signaling is upregulated, the fragment ofthe OPCML polypeptide comprises or consists of the Ig3 domain of OPCML(SEQ ID NO: 3), as discussed above.

The cancer for which the treatment regime is selected or identified maybe ovarian cancer, breast cancer, renal cancer, gastrointestinal cancer,brain cancer, lung cancer, nasopharyngeal cancer, esophageal cancer,gastric cancer, colon cancer, liver cancer, cervical cancer, prostatecancer, non-Hodgkin lymphoma, Hodgkin lymphoma, and nasal NK/T-celllymphoma. Preferably, the cancer is ovarian cancer or breast cancer.

A sixth aspect of the invention provides a method for selecting atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is HER2+; and    -   selecting a treatment regime that includes administering to the        patient a compound comprising or consisting of an OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof which        comprises the Ig3 domain of OPCML (SEQ ID NO: 3), or a variant        thereof having at least 90% sequence identity with the OPCML        polypeptide or the fragment thereof, or a nucleic acid molecule        which encodes the OPCML polypeptide or fragment or variant        thereof, and a HER2 inhibitor.

This aspect of the invention provides a method for selecting a treatmentregime for a cancer patient, the method comprising:

-   -   determining whether the cancer is HER2+; and    -   selecting a treatment regime that includes administering to the        patient a compound comprising or consisting of an OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof which        comprises the Ig3 domain of OPCML (SEQ ID NO: 3), or a variant        thereof having at least 90% sequence identity with the OPCML        polypeptide or the fragment thereof, or a nucleic acid molecule        which encodes the OPCML polypeptide or fragment or variant        thereof, and a HER2 inhibitor.

Thus, this aspect of the invention provides a method for identifying atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is HER2+ or        determining whether the cancer is HER2+;    -   wherein a HER2+ cancer indicates that a treatment regime that        includes administering a compound comprising or consisting of an        OPCML polypeptide (SEQ ID NO: 1), or a fragment thereof which        comprises the Ig3 domain of OPCML (SEQ ID NO: 3), or a variant        thereof having at least 90% sequence identity with the OPCML        polypeptide or the fragment thereof, or a nucleic acid molecule        which encodes the OPCML polypeptide or fragment or variant        thereof, and a HER2 inhibitor, will be more effective than a        treatment regime that does not include administering the OPCML        polypeptide or fragment or variant or nucleic acid molecule.

Determining whether or not a cancer is HER2+ can be carried out usingmethods well known in the art and described herein.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, for theHER2 inhibitor, and for formulations and routes of administration, areas described above in the first aspect of the invention.

The HER2+ cancer for which the treatment regime is selected oridentified may be ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, colon cancer, liver cancer,cervical cancer, prostate cancer, non-Hodgkin lymphoma, Hodgkinlymphoma, and nasal NK/T-cell lymphoma. Preferably, the cancer isovarian cancer or breast cancer.

A seventh aspect of the invention provides a method for selecting atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is OPCML+ and HER2+;        and    -   selecting a treatment regime that includes administering to the        patient a HER2 inhibitor.

This aspect of the invention provides a method for selecting a treatmentregime for a cancer patient, the method comprising:

-   -   determining whether the cancer is OPCML+ and HER2+; and    -   selecting a treatment regime that includes administering to the        patient a HER2 inhibitor.

Thus, this aspect of the invention provides a method for identifying atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is OPCML+ and HER2+,        or determining whether the cancer is OPCML+ and HER2+;    -   wherein a OPCML+ and HER2+ cancer indicates that a treatment        regime that includes administering a HER2 inhibitor will be more        effective than for a patient who has a cancer that is HER2+ and        OPCML negative.

Determining whether or not a cancer is OPCML+ and HER2+ can be carriedout using methods well known in the art and described herein.

Preferences for the HER2 inhibitor, and for formulations and routes ofadministration, are as described above in the first aspect of theinvention.

The OPCML+ and HER2+ cancer for which the treatment regime is selectedor identified may be ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, colon cancer, liver cancer,cervical cancer, prostate cancer, non-Hodgkin lymphoma, Hodgkinlymphoma, and nasal NK/T-cell lymphoma. Preferably, the cancer isovarian cancer or breast cancer.

In an embodiment, the selected treatment further comprises administeringto the patient compound comprising or consisting of an OPCML polypeptide(SEQ ID NO: 1), or a fragment thereof which comprises the Ig3 domain ofOPCML (SEQ ID NO: 3), or a variant thereof having at least 90% sequenceidentity with the OPCML polypeptide or the fragment thereof, or anucleic acid molecule which encodes the OPCML polypeptide or fragment orvariant thereof. Preferences for the compound are as described above inthe first aspect of the invention.

An eighth aspect of the invention provides a method for selecting atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is OPCML+ and        FGFR1+; and    -   selecting a treatment regime that includes administering to the        patient an FGFR1 inhibitor.

This aspect of the invention provides a method for selecting a treatmentregime for a cancer patient, the method comprising:

-   -   determining whether the cancer is OPCML+ and FGFR1+; and    -   selecting a treatment regime that includes administering to the        patient an FGFR1 inhibitor.

Thus, this aspect of the invention provides a method for identifying atreatment regime for a cancer patient, the method comprising:

-   -   identifying a patient having a cancer which is OPCML+ and        FGFR1+, or determining whether the cancer is OPCML+ and FGFR1+;    -   wherein a OPCML+ and FGFR1+ cancer indicates that a treatment        regime that includes administering an FGFR1 inhibitor will be        more effective than for a patient who has a cancer that is        FGFR1+ and OPCML negative.

Determining whether or not a cancer is OPCML+ and FGFR1+ can be carriedout using methods well known in the art and described herein.

Preferences for the FGFR1 inhibitor, and for formulations and routes ofadministration, are as described above in the first aspect of theinvention.

The OPCML+ and FGFR1+ cancer for which the treatment regime is selectedor identified may be ovarian cancer, breast cancer, renal cancer,gastrointestinal cancer, brain cancer, lung cancer, nasopharyngealcancer, esophageal cancer, gastric cancer, colon cancer, liver cancer,cervical cancer, prostate cancer, non-Hodgkin lymphoma, Hodgkinlymphoma, and nasal NK/T-cell lymphoma. Preferably, the cancer isovarian cancer or breast cancer.

In an embodiment, the selected treatment further comprises administeringto the patient compound comprising or consisting of an OPCML polypeptide(SEQ ID NO: 1), or a fragment thereof which comprises the Ig2 domain ofOPCML (SEQ ID NO: 2), or a variant thereof having at least 90% sequenceidentity with the OPCML polypeptide or the fragment thereof, or anucleic acid molecule which encodes the OPCML polypeptide or fragment orvariant thereof. Preferences for the compound are as described above inthe first aspect of the invention.

We have also shown that the OPCML status of a cancer, alone or incombination with other genetic markers, can be used to classify andstratify the cancer, and to predict the progression free survival (PFS)of cancer patients. We have shown that patients with ovarian, breast,lung and brain cancers that have relatively high levels of OPCML (i.e.,above-median levels) have an increased PFS compared to those patientswith relatively low levels of OPCML (i.e., below median levels).

Accordingly, the invention provides for the use of means for determiningthe level of OPCML in a method of predicting PFS of the cancer patient.Many suitable means are described herein. The invention also includesmethods of predicting PFS of the cancer patient characterized in thatOPCML levels in the cancer are determined.

A ninth aspect of the invention provides a method of predicting anincreased progression free survival (PFS) in a patient with ovariancancer, the method comprising:

-   -   determining, from a suitable sample obtained from the patient,        the level of OPCML and HER2 in the cancer,    -   wherein an above-median level of OPCML and an upper-quartile        HER2 level is indicative of an increased PFS.

This aspect of the invention provides for the use of means fordetermining the level of OPCML and HER2 in a method of predicting PFS ofan ovarian cancer patient. Many suitable means are described herein. Theinvention also includes methods of predicting PFS of an ovarian cancerpatient characterized in that OPCML and HER2 levels in the cancer aredetermined.

A tenth aspect of the invention provides a method of predicting anincreased PFS in a patient with lung cancer, the method comprising:

-   -   determining, from a suitable sample obtained from the patient,        the level of OPCML and the presence of EGFR mutations in the        cancer,    -   wherein an above-median OPCML level and the presence of EGFR        mutations is indicative of an increased PFS.

Suitable EGFR mutations include those described, for example, in Kosakaet al (2004) “Mutations of the epidermal growth factor receptor gene inlung cancer: biological and clinical implications” Cancer Res. 64(24):8919-23.

This aspect of the invention provides for the use of means fordetermining the level of OPCML and means for identifying EGFR mutationsin a method of predicting PFS of a lung cancer patient. Many suitablemeans are described herein and known in the art. The invention alsoincludes methods of predicting PFS of a lung cancer patientcharacterized in that OPCML levels and EGFR mutations in the cancer aredetermined.

An eleventh aspect of the invention provides a method of predicting anincreased PFS in a patient with breast cancer, the method comprising:

-   -   determining, from a suitable sample obtained from the patient,        the level of OPCML, HER2 and EGFR in the cancer,    -   wherein an above median-level of OPCML, and HER2 and EGFR levels        within the 3 uppermost quartiles (i.e., excluding the lowermost        quartile), is indicative of an increased PFS.

This aspect of the invention provides for the use of means fordetermining the level of OPCML, HER2 and EGFR in a method of predictingPFS of a breast cancer patient. Many suitable means are describedherein. The invention also includes methods of predicting PFS of abreast cancer patient characterized in that OPCML, HER2 and EGFR levelsin the cancer are determined.

A twelfth aspect of the invention provides a method of predicting anincreased PFS in a patient having glioma, the method comprising:

-   -   determining, from a suitable sample obtained from the patient,        the level of OPCML in the cancer,    -   wherein an above-median level of OPCML in the cancer is        indicative of an increased PFS.

In an embodiment, an upper-quartile level of OPCML is indicative of anincreased PFS in a patient having glioma.

This aspect of the invention provides for the use of means fordetermining the level of OPCML in a method of predicting PFS of apatient with glioma. Many suitable means are described herein. Theinvention also includes methods of predicting PFS of a patient havingglioma characterized in that OPCML levels in the cancer are determined.

A thirteenth aspect of the invention provides a method of predicting anincreased PFS in a patient having HER2+ cancer, the method comprising:

-   -   determining, from a suitable sample obtained from the patient,        the level of OPCML in the cancer,    -   wherein an upper-quartile level of OPCML in the patient sample        is indicative of an increased PFS.

Typically, the HER2+ cancer is selected from ovarian cancer, breastcancer, lung cancer and gastric cancer. Most preferably, the cancer isovarian cancer.

This aspect of the invention provides for the use of means fordetermining the level of OPCML in a method of predicting PFS of apatient with HER2+ cancer. Many suitable means are described herein. Theinvention also includes methods of predicting PFS of a patient havingHER2+ cancer characterized in that OPCML levels in the cancer aredetermined.

Typically, in the ninth to thirteenth aspects of the invention, thesuitable sample may be a cancer sample from the patient. In anembodiment of these aspects of the invention, the method may furthercomprise the prior step of obtaining the suitable sample from thepatient.

In those aspects where OPCML, HER2 and/or EGFR levels are determined, itis appreciated that the polypeptide levels may be determined. This maybe measured using methods such as IHC, or by newer methods such asreverse phase protein array (RPPA) which is a high-throughput proteomicstechnology (see, for example, Sheehan et al (2005) “Use of Reverse PhaseProtein Microarrays and Reference Standard Development for MolecularNetwork Analysis of Metastatic Ovarian Carcinoma” Molecular & CellularProteomics 4: 346-355).

Additionally or alternatively, in those aspects where OPCML, HER2 and/orEGFR levels are determined, it is appreciated mRNA levels may bedetermined (either directly or via cDNA). This can be carried out bymethods such as qRT-PCR, or microarray expression.

Typically, the level of OPCML, HER2 and/or EGFR is determined incomparison to a population of individuals having cancer in the sametissue or organ as the patient.

In another embodiment of the ninth to thirteenth aspects of theinvention, the OPCML levels can be measured by determining loss ofheterozygosity (LOH), or gene methylation. As is now well known,methylation of OPCML lowers the expression and activity of OPCML. Thus,increased methylation correlates with decreased OPCML mRNA expressionand protein activity.

We have demonstrated correlations and interactions between OPCML andHER2, HER4, FGFR1 and FGFR3, and have identified new and usefulcombinations of agents that have utility in cancer research, analysisand treatment.

Accordingly, a fourteenth aspect of the invention provides a kit ofparts comprising reagents for measuring OPCML levels and reagents formeasuring HER2 and/or FGFR1 levels.

The kit may comprise oligonucleotides for the amplification of OPCMLpolynucleotides and oligonucleotides for the amplification of HER2and/or FGFR1 polynucleotides. In this embodiment, the kit may furthercomprise reagents for the preparation of RNA from a tissue sample,and/or reagents for transcribing cDNA from mRNA, which are well known inthe art.

The kit may comprise nucleic acid molecules that specifically hybridizeto the OPCML gene and which are suitable for use as an in situhybridization probe and nucleic acid molecules that specificallyhybridize to the HER2 and/or FGFR1 gene and which are suitable for useas an in situ hybridization probe. In this embodiment, the kit mayfurther comprise reagents for individually labeling the nucleic acidmolecules and/or reagents for individually detecting the nucleic acidmolecules, which are well known in the art.

The kit may comprise antibodies that specifically bind to OPCMLpolypeptide and antibodies that specifically bind to HER2 and/or FGFR1polypeptide. In this embodiment, the kit may further comprise reagentsfor individually labeling the antibodies and/or reagents forindividually detecting the antibodies, which are well known in the art.

In these kit embodiments and aspects of the invention, the kit typicallyalso comprises a sterile container which contains the variouscomponents; such containers can be boxes, ampoules, bottles, vials,tubes, bags, pouches, blister-packs, or other suitable container formsknown in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingresearch reagents and medicaments.

A fifteenth aspect of the invention provides a kit of parts, comprisingreagents for detecting upregulation, e.g., the presence or expression,of HER2, HER4, FGFR1, EPHA2 and/or FGFR3; and a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofcomprising at least one Ig domain of OPCML, or a variant thereof havingat least 90% sequence identity with the OPCML polypeptide or thefragment thereof, or a nucleic acid molecule which encodes the OPCMLpolypeptide or fragment or variant thereof.

The reagents for detecting upregulation of HER2, HER4, FGFR1, EPHA2and/or FGFR3 may comprise antibodies that specifically bind to HER2,HER4, FGFR1, EPHA2 and/or FGFR3 polypeptide, or oligonucleotides for theamplification of HER2, HER4, FGFR1, EPHA2 and/or FGFR3 polynucleotides,or nucleic acid molecules that specifically hybridize to the HER2, HER4,FGFR1, EPHA2 and/or FGFR3 gene and which are suitable for use as an insitu hybridization probe. Many suitable reagents are well known in theart and are commercially available.

The kits may further comprise reagents for the preparation of RNA from atissue sample and/or reagents for transcribing cDNA from mRNA, reagentsfor individually labeling the nucleic acid molecules for use ashybridization probes and/or reagents for individually detecting thenucleic acid molecules, or reagents for individually labeling theantibodies and/or reagents for individually detecting the antibodies.

In a specific embodiment, the kit comprises reagents for detectingupregulation, e.g., the presence or expression, of HER2, and a compoundcomprising or consisting of an OPCML polypeptide (SEQ ID NO: 1), or afragment thereof comprising at least the Ig3 domain of OPCML (SEQ ID No:3), or a variant thereof having at least 90% sequence identity with theOPCML polypeptide or the fragment thereof, or a nucleic acid moleculewhich encodes the OPCML polypeptide or fragment or variant thereof.

In another specific embodiment, the kit comprises reagents for detectingupregulation, e.g., the presence or expression, of FGFR1; and a compoundcomprising or consisting of an OPCML polypeptide (SEQ ID NO: 1), or afragment thereof comprising at least the Ig2 domain of OPCML (SEQ ID No:2), or a variant thereof having at least 90% sequence identity with theOPCML polypeptide or the fragment thereof, or a nucleic acid moleculewhich encodes the OPCML polypeptide or fragment or variant thereof.

Optionally, the fragments of the OPCML polypeptide or variant thereoffurther comprises the Ig1 domain of OPCML (SEQ ID NO: 4) or may be fusedto an antibody Fc fragment.

All further preferences for the compound comprising or consisting of anOPCML polypeptide, or the fragment or variant thereof, or the nucleicacid molecule which encodes the polypeptide or fragment or variant, andfor suitable formulations thereof, are as described above in the firstaspect of the invention.

A sixteenth aspect of the invention provides a kit of parts, comprisinga compound comprising or consisting of an OPCML polypeptide (SEQ ID NO:1), or a fragment thereof which comprises the Ig3 domain of OPCML (SEQID No: 3), or a variant thereof having at least 90% sequence identitywith the OPCML polypeptide or the fragment thereof, or a nucleic acidmolecule which encodes the OPCML polypeptide or fragment or variantthereof; and a HER2 inhibitor.

Optionally, the fragments of the OPCML polypeptide or the variantthereof further comprises the Ig1 domain of OPCML (SEQ ID NO: 4) or maybe fused to an antibody Fc fragment.

Optionally, the kit further comprises reagents for detecting thepresence or expression of HER2 as described above. Additionally oralternatively, the kit may further comprise reagents for measuring OPCMLlevels as described above.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe HER2 inhibitor, as well as suitable formulations thereof, are asdescribed above in the first aspect of the invention.

A seventeenth aspect of the invention provides a kit of parts,comprising a compound comprising or consisting of an OPCML polypeptide(SEQ ID NO: 1), or a fragment thereof which comprises the Ig2 domain ofOPCML (SEQ ID No: 2), or a variant thereof having at least 90% sequenceidentity with the OPCML polypeptide or the fragment thereof, or anucleic acid molecule which encodes the OPCML polypeptide or fragment orvariant thereof; and an FGFR1 inhibitor.

Optionally, the fragments of the OPCML polypeptide or the variantthereof further comprise the Ig1 domain of OPCML (SEQ ID NO: 4) or maybe fused to an antibody Fc fragment.

Optionally, the kit further comprises reagents for detecting thepresence or expression of FGFR1 as described above. Additionally oralternatively, the kit may further comprise reagents for measuring OPCMLlevels as described above.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe FGFR1 inhibitor, as well as suitable formulations thereof, are asdescribed above in the first aspect of the invention.

An eighteenth aspect of the invention provides a kit of parts,comprising a compound comprising or consisting of an OPCML polypeptide(SEQ ID NO: 1), or a fragment thereof which comprises at least one Igdomain of OPCML, or a variant thereof having at least 90% sequenceidentity with the OPCML polypeptide or the fragment thereof, or anucleic acid molecule which encodes the OPCML polypeptide or fragment orvariant thereof; and a platinum based chemotherapeutic agent.

Optionally, the fragments of the OPCML polypeptide of the variantthereof further comprises the Ig1 domain of OPCML (SEQ ID NO: 4) or maybe fused to an antibody Fc fragment.

Optionally, the kit may further comprise reagents for measuring OPCMLlevels as described above.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe platinum based chemotherapeutic agent, as well as suitableformulations thereof, are as described above in the first aspect of theinvention.

A nineteenth aspect of the invention provides a pharmaceuticalcomposition comprising a compound comprising or consisting of an OPCMLpolypeptide (SEQ ID NO: 1), or a fragment thereof which comprises theIg3 domain of OPCML (SEQ ID No: 3), or a variant thereof having at least90% sequence identity with the OPCML polypeptide or the fragmentthereof, or a nucleic acid molecule which encodes the OPCML polypeptideor fragment or variant thereof, and a HER2 inhibitor.

The invention also includes the combination of a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises the Ig3 domain of OPCML (SEQ ID No: 3), or a variantthereof having at least 90% sequence identity with the OPCML polypeptideor the fragment thereof, or a nucleic acid molecule which encodes theOPCML polypeptide or fragment or variant thereof, and a HER2 inhibitor,for use in medicine.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe HER2 inhibitor, as well as suitable formulations thereof, are asdescribed above in the first aspect of the invention.

A twentieth aspect of the invention provides a pharmaceuticalcomposition comprising a compound comprising or consisting of an OPCMLpolypeptide (SEQ ID NO: 1), or a fragment thereof which comprises theIg2 domain of OPCML (SEQ ID No: 2), or a variant thereof having at least90% sequence identity with the OPCML polypeptide or the fragmentthereof, or a nucleic acid molecule which encodes the OPCML polypeptideor fragment or variant thereof, and an FGFR1 inhibitor.

The invention also includes the combination of a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises the Ig2 domain of OPCML (SEQ ID No: 2), or a variantthereof having at least 90% sequence identity with the OPCML polypeptideor the fragment thereof, or a nucleic acid molecule which encodes theOPCML polypeptide or fragment or variant thereof, and an FGFR1inhibitor, for use in medicine.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe FGFR1 inhibitor, as well as suitable formulations thereof, are asdescribed above in the first aspect of the invention.

A twenty-first aspect of the invention provides a pharmaceuticalcomposition comprising a compound comprising or consisting of an OPCMLpolypeptide (SEQ ID NO: 1), or a fragment thereof which comprises atleast one Ig domain of OPCML, or a variant thereof having at least 90%sequence identity with the OPCML polypeptide or the fragment thereof, ora nucleic acid molecule which encodes the OPCML polypeptide or fragmentor variant thereof, and a platinum based chemotherapeutic agent.

The invention also includes the combination of a compound comprising orconsisting of an OPCML polypeptide (SEQ ID NO: 1), or a fragment thereofwhich comprises at least one Ig domain of OPCML, or a variant thereofhaving at least 90% sequence identity with the OPCML polypeptide or thefragment thereof, or a nucleic acid molecule which encodes the OPCMLpolypeptide or fragment or variant thereof, and a platinum basedchemotherapeutic agent, for use in medicine.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, and forthe platinum based chemotherapeutic agent, as well as suitableformulations thereof, are as described above in the first aspect of theinvention.

A twenty-second aspect of the invention provides a method of testing atreatment regime for efficacy in treating cancer, the method comprising:

-   -   providing a first group of individuals with cancer;    -   administering a test treatment regime to the first group of        individuals, wherein the test treatment regime comprises        administering a compound comprising or consisting of OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof comprising at        least one Ig domain of OPCML, or a variant thereof having at        least 70% sequence identity with the OPCML polypeptide or the        fragment thereof, or a nucleic acid molecule which encodes the        OPCML polypeptide or fragment or variant thereof;    -   comparing the first group of individuals to a control group of        individuals; and    -   assessing whether the treatment regime has contributed to        treating the cancer in the a first group of individuals.

A related aspect of the invention provides a method of comparingtreatment regimes for the treatment of cancer, the method comprising:

-   -   providing first and second groups of individuals with cancer;    -   administering a test treatment regime to the first group of        individuals, wherein the test treatment regime comprises        administering a compound comprising or consisting of OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof comprising at        least one Ig domain of OPCML, or a variant thereof having at        least 70% sequence identity with the OPCML polypeptide or the        fragment thereof, or a nucleic acid molecule which encodes the        OPCML polypeptide or fragment or variant thereof;    -   administering a second treatment regime to the second group of        individuals;    -   comparing the first and second groups of individuals; and    -   assessing whether the test treatment regime has improved the        treatment of the cancer in the first group of individuals        compared to the treatment of the cancer second group of        individuals.

As is well known in the art, to control for the ‘placebo effect’, it maybe desirable to substitute the test treatment regime for a placebo in aproportion of the first and/or second group of individuals undergoingthe assessment.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, as wellas suitable formulations thereof, are as described above in the firstaspect of the invention.

In the embodiment where the method is conducted in the context of aclinical trial, the individuals with cancer may be human patients withcancer: The human patient may have a cancer selected from ovariancancer, breast cancer, renal cancer, gastrointestinal cancer, braincancer, lung cancer, nasopharyngeal cancer, esophageal cancer, gastriccancer, colon cancer, liver cancer, cervical cancer, prostate cancer,non-Hodgkin lymphoma, Hodgkin lymphoma, and nasal NK/T-cell lymphoma. Itis preferred that the cancer is ovarian cancer or breast cancer.

In another embodiment, the individuals with cancer may be non-humananimal models of cancer. The animal model of cancer may be a model ofovarian, lung, breast, colorectal, pancreatic, gastric, esophageal,renal, biliary, hepatic, cervical, uterine or prostate cancer. Theanimal model of cancer may be a xenograph model of cancer. Typically,the animal model of cancer is a mouse model of cancer, which may be nudemouse, scid mouse or oncomouse model of cancer.

In an embodiment, the test treatment regime is administered byintravenous or intraperitoneal administration.

Assessment of the test treatment regime may be carried out multipletimes, for example at regular intervals such as weekly, monthly, everysix months or every year in order to monitor efficacy of the testtreatment over time.

Assessment of the test treatment regime may suitably comprise PK and/orPD analysis of the first group of individuals or in vivo molecularimaging of the first group of individuals using FLT, FDG, aposense orPET tracers.

In an embodiment, assessment of the individuals may include assessing,in a suitable sample obtained from the individuals, the level of DNAmethylation of the OPCML gene and/or OPCML loss of heterozygosity.

In an additional or alternative embodiment, assessment of theindividuals may include assessing, in a suitable sample obtained fromthe individuals, mRNA or protein expression levels and/or gene copynumber of FGFR1, FGFR3, HER2, HER4 and/or EGFR.

Typically, the compound comprising or consisting of the OPCMLpolypeptide or the fragment or variant thereof is detectably labeled forin vivo imaging.

In an embodiment, the test treatment regime further comprisesadministration of an FGFR1 inhibitor, an FGFR3 inhibitor, an HER2inhibitor, an HER4 inhibitor, an EPHA2 inhibitor and/or an EGFRinhibitor to the first group of individuals. Suitable inhibitors arewell known in the art and described herein. Preferably, the HER2inhibitor is an anti-HER2 antibody such as trastuzumab or pertuzumab, oris selected from the group consisting of lapatinib, neratinib and BIBW2992. Also preferably, the FGFR1 inhibitor is selected from the groupconsisting of PD 173074, SU5402 and Indirubin-3′-monoxime.

Additionally or alternatively, the test treatment regime may furthercomprise administration of a platinum based chemotherapeutic agent tothe first group of individuals.

Preferences for the FGFR1, FGFR3, HER2, HER4, EPHA2 and/or EGFRinhibitors, and for the platinum based chemotherapeutic agents, as wellas suitable formulations thereof, are as described above in the firstaspect of the invention.

Additionally or alternatively, the test treatment regime may yet furthercomprise radiotherapy of the first group of individuals.

A twenty-third aspect of the invention provides a method of identifyingan agent that may be useful in the treatment of cancer, or a leadcompound for the identification of an agent that may be useful in thetreatment of cancer, the method comprising:

-   -   providing a test agent comprising or consisting of OPCML        polypeptide (SEQ ID NO: 1), or a fragment thereof comprising at        least one Ig domain of OPCML, or a variant thereof having at        least 70% sequence identity with the OPCML polypeptide or the        fragment thereof, or a nucleic acid molecule which encodes the        OPCML polypeptide or fragment or variant thereof; and    -   testing the candidate agent in an in vitro anti-cancer assay,    -   wherein a candidate agent that shows efficacy in the anti-cancer        assay may be a compound that is useful in the treatment of        cancer, or may be a lead compound for the identification of an        agent that is useful in the treatment of cancer.

Preferences for the compound comprising or consisting of an OPCMLpolypeptide, or the fragment or variant thereof, or the nucleic acidmolecule which encodes the polypeptide or fragment or variant, as wellas suitable formulations thereof, are as described above in the firstaspect of the invention. Thus, for example, the OPCML polypeptide may berecombinant human OPCML (SEQ ID No: 1); the OPCML polypeptide fragmentmay comprise or consist of the Ig2 domain of human OPCML (SEQ ID No: 2);the OPCML polypeptide fragment comprises may comprise or consist of theIg3 domain of human OPCML (SEQ ID No: 3); and the fragment of the OPCMLpolypeptide may further comprises the Ig1 domain of human OPCML (SEQ IDNO: 4) or may be fused to an antibody Fc fragment.

The variant of the OPCML polypeptide or of the fragment thereoftypically has at least 80% sequence identity, or at least 85%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95% or at least 96%, or at least 97%, or at least98%, or at least 99% sequence identity, with the human OPCML polypeptide(SEQ ID No:1) or the fragment thereof.

The in vitro anti-cancer assay may be a cellular proliferation assay ofprimary cancer cells or cancer cell lines in vitro, such as described inthe examples and well known in the art. Other suitable assays includeassays for RTK downregulation, and downregulation ofphosphoERK/PhosphoAKT as are known in the art.

In an embodiment, the identified compound is modified, and the modifiedcompound is tested for efficacy in an in vitro anti-cancer assay.Further, the identified compound or the modified compound is tested forefficacy in an animal model, typically a mouse model, of cancer.

The method may further comprise the step of synthesising, purifyingand/or formulating the identified compound or the modified compound.

The invention will now be described in more detail with respect to thefollowing Figures and Examples.

FIG. 1: Expression of OPCML is induced by EGF and FGF1/2. (A) OPCMLinduction by EGF and FGF1/2 in SK-OV-3: (i) induction of OPCMLexpression upon EGF stimulation (50 ng/ml) determined by qRT-PCR; (ii)induction of OPCML protein under same conditions. (B) (i) Induction ofOPCML expression by FGF1 and FGF2 stimulation (black and grey linesrespectively) at 10 ng/ml; (ii) induction of the OPCML protein under thesame FGF2 conditions.

FIG. 2: OPCML interacts with the ECD of HER2 and FGFR1 but not EGFR. (A)(i): Co-IP was performed using protein extracts from SKOBS-V1.2,SKOBS-3.5 and BKS2.1 (SKOBS-3.5 and BKS2.1 express 3 and 20-fold moreOPCML protein than normal physiological levels seen in OSE-C2). The goatpolyclonal anti-OPCML (R & D Systems) was used in initial IP andproducts were detected on Western blots employing respective Abs forEGFR and HER2. The reciprocal IP with a monoclonal to ECD of HER2(Calbiochem) is shown with successful immunoblotting for OPCML in theBKS2.1 stable line (expressing 20-fold more OPCML); (ii) Co-IP using theOPCML Ab in SKOBS-V1.2 and BKS2.1 lines were immunoblotted withanti-FGFR1αECD Ab. (B) A structural representation of GST-OPCML 1+2 andGST-OPCML 1+2+3 constructs with subsequent Western blot analysis of thefusion proteins. (C) (i) GST pull-down assay with SK-OV-3 cell lysateshowing binding of HER2 to GST-OPCML 1+2+3 only (lane 2), and no bindingof EGFR to any GST-OPCML protein (top panel). FGFR1 was also shown tobind GST-OPCML 1+2+3 and GST-OPCML 1+2 from the same pull-down withlysate from SK-OV-3 cells transiently transfected with FGFR1 cDNA. Thepresence of the OPCML fusion proteins in these assays was verified byimmunoblotting (bottom panel); (ii) In vitro interactions wereundertaken verifying binding of HER2 and FGFR1 extracellular domainprotein generated from in vitro transcription and translation kit (TnT)to GST-OPCML fusion proteins. HER2ECD is seen to bind to GST-OPCML 1+2+3only suggesting that domain 3 is critical whereas FGFR1 binds to GSTOPCML 1+2 suggesting binding outwith domain 3.

FIG. 3: Expression of OPCML in ovarian cancer cells downregulates HER2and FGFR1 but not EGFR or FGFR2, abrogates HER2, EGFR and FGFR1 tyrosinephosphorylation, and reduces growth. (A) (i) Stable OPCML results in theabrogation of HER2 protein expression, but not EGFR protein expression;(ii) Similarly, OPCML stable expression leads to downregulated FGFR1 butnot FGFR2. (B) (i) Immunofluorescence microscopy showing HER2 expressionin control SKOBS-V1.2 and OPCML expressing BKS2.1 cells. Abundant HER2expression seen in SKOBS-V1.2 is reduced in BKS2.1 line associated withexpression of OPCML. Note co-localisation of OPCML and HER2; (ii) chartillustrating the mean pixel intensity of receptor tyrosine kinases HER2,EGFR, FGFR1 FGFR2 in the context of minimal (SKOBS 2.1) or high (BKS2.1)OPCML expression showing significant downregulation of HER2 and FGFR1.(C) (i) Phosphorylation of specific key tyrosines was analysed bywestern blotting in BKS2.1 compared to the SKOBS-V1.2 line usingphospho-antibodies. Phospho-EGFR Y1173, phospho-HER2 Y1248 andphospho-FGFR1 Y766 were all profoundly abrogated with expression ofOPCML; (ii) Similar analysis of downstream signaling adaptor andsubstrates also exhibited strong downregulation of phospho signal atPLCγ, Grb2 and ERKp42/p44 in response to OPCML expression/EGFStimulation; (iii) Western analysis of phosphorylated Akt (Ser 473 andThr 308) as well as total Akt protein in SKOBS-V1.2 and BKS2.1expressing high levels of OPCML; (iv) proliferation curves of OPCMLstable clones (BKS2.1 and SKOBS-3.5), and OSE-C2 cells compared to theSK-OV-3 empty vector control line (SKOBS-V1.2) on 0.25% FCS-supplementedmedium with 50 ng/ml EGF as measured by MTT assay. (D) (i) Expression ofOPCML downregulates HER2, FGFR1 and FGFR3 expression; (ii) Completeprotein knockdown of physiological OPCML in OSE-C2 cells with no loss inthe non-silencing control (NS) results in an increase in total HER2 andFGFR1 compared with controls. (E) Examples of Immunofluorescentmicroscopy images used in the relative abundance measurement in (B),showing the relative signal intensity of EGFR and FGFR2 remainingconstant, but FGFR1 levels drastically reduced in BKS2.1.

FIG. 4: OPCML presents HER2/EGFR heterodimer formations. (A) SKOBS-V1.2and BKS2.1 cell extracts were subjected to Co-IP and immunoblotted withthe indicated antibodies, demonstrating loss of heterodimerisation. (B)Monolayers of SKOBS-V1.2, SKOBS3.5 and BKS2.1 were incubated with 0.02pg/ml [¹²⁵1] EGF for 2 h at 4° C. Levels of radiolabel led EGF bindingwere determined by detection of gamma emission.

FIG. 5: OPCML localizes in lipid rafts, and enhances HER2 ubiquitinationand proteasomal degradation. (A) (i) OPCML localizes to detergentinsoluble fraction indicating location in lipid rafts (R—raft;NR—non-raft); (ii) Majority of internalized OPCML colocalizes withcaveolin-1 (a marker of the raft-caveolar pathway) compared to EEA-1(early endosome marker); (iii) Immunofluorescence microscopy of SK-OV-3and BKS2.1 cells growing on glass slides after being fixed andpermeabilised. OPCML and HER2 were detected in (a) SK-OV-3 ovariancancer line and (b) BKS2.1 using anti-mouse mAb OPCML and rabbit mAbHER2 antibodies and fluorescently labeled secondary antibody to murine(Alexa—594) and rabbit (Alexa—488) immunoglobulins. OPCML and EGFR weredetected in (c) SK-OV-3 and (d) BKS2.1 cells using mouse mAb OPCML andgoat pAb EGFR antibodies and fluorescently labeled secondary antibody tomurine (Alexa—594) and goat (Alexa—488) immunoglobulins. (B) (i) BKS2.1and SKOBS-V1.2 transfected with HA-tagged-ubiquitin plasmid were serumstarved then stimulated with 50 ng/ml EGF before beingimmunoprecipitated with HER2 and immunoblotted with HA Ab, highermolecular weight shift is seen with HA signifying polyubiquitination ofHER2; (ii) Densitometric analysis of HA-ubiquitin WB of HER-IP samplesfrom −1+ OPCML expressing cell lines. (C) (i)&(ii) OPCML expressioninduces a shift in proportion of HER-2. (D) (i)&((ii) SKOBS-V1.2 andBKS2.1 cells were treated with EGF (50 ng/ml) for 60 minutes in thepresence or absence of the proteasomal inhibitor MG-132 (1 μm). Celllysates were subjected to immunoblotting with the indicated antibodies.Ablation of HER2 protein level in the BKS2.1 line is restored in thepresence of MG-132 whereas in SKOBS-V1.2 both +/−MG-132 lanes have equalintensity HER2 immunoreactivity. The lysates were also challenged withFGFR1-specific Abs showing similar OPCML-specific upregulation of the 79kD FGFR1 band in the absence of MG-132; (iii) Disruption of cholesterolusing methyl-β-cyclodextrin inhibits degradation of HER2.

FIG. 6: The sensitivity to the dual HER2-EGFR specific TKI lapatinib andHER2-specific trastuzumab. (A) SKOBS-V1.2 and BKS2.1 cells werestimulated with 50 ng/ml EGF for 30 minutes in absence/presence of 1, 2and 10 nM lapatinib (i) or 5 and 10 μg/ml trastuzumab (ii) and blottedfor phospho-ERK and phospho-Akt. The extent of pERK inhibition bylapatinib was confirmed by densitometry (data not shown). (B) The effectof OPCML knockdown on the efficiency of lapatinib-induced pERKattenuation was analysed; densitometry of pERK signal (mean values of 3independent experiments). Lapatinib reduced the EGF-induced pERK signalby 70% in the presence of non-silencing (non-si) duplex. Knockdown bysiRNA to OPCML abrogated the lapatinib mediated inhibition of pERKsignal significantly, reducing the extent of inhibition to 47%(P=0.0009). (C) The sensitization of ovarian cancer cell lines(SKOBS-3.5 and BKS2.1) to lapatinib compared to SKOBS-V1.2 illustratesBKS2.1 achieving a >10-fold increase in sensitivity. The extent of pERKinhibition by lapatinib is illustrated in the densitometry bar chartdepicting the % intensity of phospho-ERK p42/42 signal after lapatinibtreatment compared to the EGF stimulation with no lapatinib administeredtaken as 100% (not shown). From the densitometry of phospho-ERK signalstrength, 1 nM lapatinib reduces the phospho-ERK signal intensity morethan 10 nM lapatinib in SKOBS-V1.2.

FIG. 7: Kaplan-Meier curves of ovarian cancer progression free survivalagainst time. (i) Survival compared between low (N=122) and high (N=129)OPCML expression (N=251 patients, RNA expression data from large Bowtelldataset). (ii) Survival between top quartile (High Her2/High OPCML[N=42] and High Her2/Low OPCML [N=17]) HER2 RNA expression and bottomquartile (Low Her2/High OPCML [N=21] and Low Her2/Low OPCML [N=41]) HER2dichotomised around OPCML median.

FIG. 8: Purification of recombinant human OPCML expressed in E. coli.(A) Schematic diagram of OPCML and the 3 Ig-domains subcloned forrecombinant expression. (B) SDS-PAGE analysis of recombinant human OPCMLexpressed in E. coli. Protein expression was induced by the addition ofIPTG (1 mM final concentration). Lane M shows protein molecular massmarkers; lane 1 shows uninduced whole cell lysate (WC); lanes 2 and 3show soluble (sol) and insoluble (ins) fraction of induced cell lysate,respectively. Gels were stained with Coomassie Brilliant Blue. (C)SDS-PAGE analysis of inclusion body purification and refolding. Lane 1shows purified inclusion bodies (IB) and lane 2 shows inclusion bodiesdenatured in 8 M Urea and refolded by dialysis into PBS. Insolubleproteins were removed by centrifugation and a sample of the supernatantretained for analysis (RE). Gels were stained with Coomassie BrilliantBlue. (D) Image of OPCML crystals obtained by hanging drop at a proteinconcentration of 10 mg/ml.

FIG. 9: Cellular uptake of recombinant OPCML. SKOV-3 cells wereincubated with vehicle alone or vehicle including 2 μM OPCML for 24 h.Cells were then washed 3× with PBS and fixed with 4% PFA. Cells werethen prepared for IFM using rabbit α-HER2 mAb and mouse α-OPCML mAb.Goat α-rabbit alexa-488 and α-mouse alexa-555 secondary antibodies wereused. Slides were imaged using a Leica SP5 confocal microscope.

FIG. 10: Exogenous OPCML administration inhibits RTK signaling in vitro.(A) Western Blot analysis of pHER, tHER2, tEGFR, pEGFR, tFGFR, pFGFRproteins from SKOV-3 cells untreated, treated with vehicle only or OPCML(2 μM) for 48 h. (B) Western blot analysis of pAKT, tAKT, pERK, tERKproteins from SKOV-3 cells untreated, treated with vehicle only or OPCML(2 μM) for 48 h. Protein loading was standardised by BCA-assay andβ-tubulin was used as a loading control.

FIG. 11: Exogenous OPCML administration inhibits growth of ovarian andbreast cancer cell lines. (A) SKOV-3 cells were subjected to a 48 h invitro growth assay with varying concentrations of OPCML administration.(B) A 96 h in vitro SKOV-3 growth assay with 2 μM rOPCML administered togrowth media. Time points were collected every 24 h and normalised tocontrol cells treated with vehicle only. Cell viability was quantifiedusing MTT and measured at 570 nm. (C) Upper panel—OSE-C2 and SKOV-3cells were subjected to a 48 h in vitro growth assay with varying OPCMLconcentrations (0.5, 1, 2, 5 and 10 μM); lower panel—normal ovariansurface epithelial cells (OSE-C2) and a panel of ovarian (SKOV-3, IGROV,OVISE, OVCAR-5, A2780, PEA1 and PEA2) and HER2-positive (MDA-231) andnegative (BT-474) breast cancer cells were subjected to an in vitrogrowth assay with 10 μm OPCML. Time points were collected at 24 h and 48h and cell growth was monitored to vehicle only controls. Cell viabilitywas quantified in all experiments using an MTT-based assay and measuredat 570 nm.

FIG. 12: Kaplan-Meier analysis of overall survival according to OPCMLdichotomised survival. Analyses were conducted for (A) breast cancer,(B) lung cancer, and (C&D) high grade gliomas. The Breast Cancermicroarray dataset, for the first of the three graphs, was taken fromWang et al (2005) “Gene-expression profiles to predict distantmetastasis of lymph-node-negative primary breast cancer” Lancet365(9460): 671-9. The Glioblastoma microarray dataset was taken fromRoel et al (2010) “Integrated Genomic Analysis Identifies ClinicallyRelevant Subtypes of Glioblastoma Characterized by Abnormalities inPDGFRA, IDH1, EGFR, and NF1” Cancer Cell 17(1): 98-110. The Lung cancermicroarray dataset was taken from Toshiyuki Takeuchi et al (2006)“Expression Profile-Defined Classification of Lung Adenocarcinoma ShowsClose Relationship With Underlying Major Genetic Changes andClinicopathologic Behaviors” Journal of Clinical Oncology 24(11):1679-1688.

FIG. 13: OPCML has functional characteristics of a tumour suppressorgene in vitro and in vivo. (A) Functional in vitro growth assayscomparing parent, OPCML wild-type sense-transfected (two independentcell lines) and OPCML antisense-transfected, clonal SKOV-3 cell lines.Cells were counted every third day for 12d. The values shown are themean±s.d. of triplicate assays. (B) Comparison of parent OPCMLsense-transfected (two independent cell lines) and OPCMLantisense-transfected SKOV-3 cell lines in subcutaneous tumor growthassay. (C) Tumor measurements were recorded every 7d for 28d.

FIG. 14: OPCML amino acid sequence from GenBank Accession No. NP_002536(SEQ ID NO: 1).

FIG. 15: Amino acid sequence of OPCML Ig1 (SEQ ID NO: 4)(Q14982|39-126), Ig2 (SEQ ID NO: 2) (Q14982\136-219) and Ig3 (SEQ ID NO:3) (Q14982|223-310) domains.

FIG. 16: Expression of OPCML in ovarian cancer cells downregulates aspecific repertoire of receptor tyrosine kinases. Western blotsdemonstrating that (A) OPCML-stable transfection in SKOV-3 and (B)transient transfection in PEO1 negatively regulates EphA2, FGFR1, FGFR3,HER2, and HER4, but not EphA10, FGFR2, EGFR, HER3, VEGFR1 and VEGFR3.(C) siRNA knockdown of OPCML showed a reciprocal increase in expressionof OPCML-regulated RTKs previously described in (A). Proteinconcentration of all cell lysates was quantified by BCA assay andconfirmed by western blot analysis of β-tubulin.

FIG. 17: OPCML directly interacts with EphA2, FGFR1 and HER2, but notEGFR. (A) Immuno-precipitation (IP) for OPCML showing positiveimmunoblots for EphA2, FGFR1 and HER2. Similarly, IP for each of theseproteins in turn showed positive immunoblots for OPCML. We were not ableto demonstrate co-IP with EGFR and OPCML after numerous attempts. (B) Aschematic representation of GST-OPCML domain 1-3 construct. (C).GST-OPCML 1-3 pull-down assay using SKOV-3 cell lysate showing specificbinding of HER2 and FGFR1 and no binding of EGFR. The presence of OPCMLfusion proteins in these assays was verified by immunoblotting (IB,bottom panel). (D) Confirmatory in vitro interaction studies wereundertaken verifying binding of HER2 and FGFR1 extracellular domainprotein, generated from in vitro translation kit (TnT) (HER2ECD) and inE. coli (FGFR-ECD) to GST-OPCML 1-3 fusion protein.

FIG. 18: OPCML associated RTK downregulation abrogates downstreamsignalling. (A) Western blot of total and phospho HER2 and EGFR proteinfrom SKOBS-V1.2 (vector only control) and BKS-2.1 (stable expression ofOPCML). Cells were subjected to a 60 min EGF timecourse (50 ng/ml). (B)Western blot of total and phosphor FGFR1 protein from SKOBS-V1.2 (vectoronly control) and BKS-2.1 (stable expression of OPCML). Cells weresubjected to a 60 min FGF1 timecourse (10 ng/ml). (C) Western blot ofdownstream signaling adaptor and substrates, phosphorylation levels ofPLCγ (Y783), ERK 1 & 2 (T202/T204) and Akt at Ser473 in response toOPCML expression. Cells were subjected to a 60 min EGF timecourse (50ng/ml).

FIG. 19: Mechanism of OPCML mediated RTK downregulation. (A) OPCMLsequesters RTKs in detergent-resistant membrane fraction; SKOBS-V1.2 andBKS2.1 cells were subjected to membrane fractionation to separatedetergent resistant membrane fraction (DRMF) or lipid raft fraction fromthe bulk membrane phase. An equal volume from each fraction was analysedby SDS-PAGE followed by Western blotting with anti-EGFR, anti-HER2,anti-OPCML and anti-Cav-1 antibodies. OPCML was found to be strictlylocated in the DRMF with Cav-1. Transfection of OPCML was associatedwith a shift of HER2 and EGFR to the DRMF. (B) Confocalco-immunofluorescence demonstrates co-localisation of rabbitanti-Cav-1/alexa488-conjugated anti-rabbit secondary with mouseanti-OPCML/alexa 555 anti mouse secondary, but not EEA1 suggesting thatOPCML is associated with caveolar endocytosis. (C) Quantification of thetotal number of OPCML pixels colocalised with Cav-1 and EEA-1,demonstrating that OPCML principally co-localises with Cav-1. (D)Ubiquitination status of HER2: SKOBS-V1.2 and BKS-2.1 transientlytransfected with an HA-tagged ubiquitin were treated with the MG-132then EGF or left untreated. Ubiquitylated proteins were detected byimmunoblotting (IB) with an anti-HA antibody and samples were run inparallel to probe for HER2 with a mouse anti-HER. These studiesdemonstrated that OPCML transfection is associated withpolyubiquitylation of HER2. (E) Bar chart to show the densitometricquantification of the immunoblotting seen in (D) showing that OPCMLexpressing BKS2.1 demonstrated increased HA-ubiquitin associated withHER2, not seen in OPCML (−) cells. (F) Confocal co-immunofluorescence ofHER2 colocalisation with Cav-1 and EEA-1 with quantification (G) as apercentage of the total number of HER2 pixels detected within the cells.These studies demonstrate that OPCML expressing cells are associatedwith a switch of HER2 vesicle trafficking from EEA-1 to Cav-1 mediatedendocytosis. (H) SKOBS-V1.2 and BKS-2.1 were treated with the lysosomalinhibitor chloroquine (CQ; 0.1 mM) or the proteasomal inhibitor MG-132(0.1 μM) for 2 h. Immunoblotting for EGFR, HER2, OPCML and (3-tubulinwas shown. (I) Densitometric analysis of these Western blot data forHER2 (upper chart) and EGFR (lower chart) using β-tubulin as a control.These studies demonstrate that MG132 but not CQ specifically increasesHER2 protein level demonstrating that HER2 is degraded preferentiallyvia a proteosomal mechanism via direct binding to OPCML followed bycaveolar endocytosis. In contrast, EGFR (that does not bind to OPCML) isnot regulated by this mechanism. (J) Cells were incubated in controlmedia or media supplemented with methyl-β-cyclodextrin (M-βCD; 0.2 mM)for 2 h to deplete cholesterol. Western blotting for HER2 andphosphoHER2 (Y1248) demonstrates that cholesterol disruption of lipidrafts also blocks this OPCML mediated HER2 degradation.

FIG. 20: Exogenous rOPCML administration specifically inhibits ovarian,breast and cancer cell growth in vitro, (A) OSE-C2 and SKOV-3 cells weresubjected to a 48 h in vitro growth assay with varying concentrations ofrOPCML (0.5, 1, 2, 5 and 10 μM). (B) a 96 h in vitro SKOV-3 growth assaywith 2 μM rOPCML administered to growth media. Time points werecollected every 24 h and normalised to control cells treated withvehicle only. (C) Normal ovarian surface epithelial cells (OSE-C2) and apanel of ovarian (SKOV-3, IGROV, OVISE, OVCAR-5, A2780, PEA1 and PEA2),HER2-positive (MDA-231) and negative (BT-474) breast cancer cells andnon-adherant (H69, H501 small-cell) and adherent (A549 carcinoma, HCC95non-SSLC squamous) lung cancer cells were subjected to an in vitrogrowth assay with 10 μM of rOPCML. Time points were collected at 24 and48 h and cell growth was normalised to vehicle only controls. Cellviability was quantified in all experiments using an MU-based assay andmeasured at 570 nm. Caspase cleavage-based apoptosis assays using (D)A2780 and (E) SKOV-3 were undertaken with administration of rOPCML (2, 5and 10 μM) over 24 h. Caspase cleavage was quantified using theGaspaseGlo kit and normalised to cell viability.

FIG. 21: Exogenous rOPCML administration abrogates total RTK levels andinhibits RTK signalling in vitro. (A) Western Blot analysis of pHER,tHER2, tEGFR, pEGFR, tFGFR, pFGFR proteins from SKOV-3 and A2780 cellstreated with vehicle only or OPCML (2 μM) for 48 h. (B) Western blotanalysis of pAKT, tAKT, pERK, tERK, proteins from SKOV-3 and A2780 cellstreated with vehicle only or OPCML (2 μM) for 48 h. Protein loading wasstandardised by BCA-assay and β-tubulin was used as a loading control.

FIG. 22: Recombinant OPCML treatment reduces tumour growth in vivo.Comparison of r-OPCML and control protein (bovine serum albumen) treatedA2780 and SKOV-3 cell lines. (A) Gross anatomical observation of theanimals (B) Intraperitoneal tumour growth assay. Tumours were removed,weighed and photographed after 3 weeks of twice-weekly 1 mLintraperitoneal injection of 10 μM r-OPCML or BSA. Representative A2780and SKOV-3 tumour xenografts from 4 animals are presented. A scale baris shown. (C) mean tumour weight, (D) number of deposits and (E) volumeof ascites removed per mouse from the intraperitoneal assay presented inB. Comparison is shown between tumours and ascites collected from BSA-(−) and r-OPCML-treated (+) mice. (F) Western blot analysis of recoveredtumours from control (BSA) and r-OPCML treated animals recapitulatesin-vitro findings.

Example 1: Opcml Tumor Suppressor Functions as a Repressor-Adaptor,Negatively Regulating Receptor Tyrosine Kinases in Both Normal OvarianSurface Epithelium and Ovarian Cancer

Summary

Epithelial ovarian cancer (EOC) is the leading cause of death fromgynecologic malignancy. Its molecular basis is poorly understood butinvolves dysfunction of p53 (Hall et al (2004) “Critical evaluation ofp53 as a prognostic marker in ovarian cancer”. Expert Reviews inMolecular Medicine 6: 1-20), BRCA1 and -2 (Radice (2002) “Mutations ofBRCA genes in hereditary breast and ovarian cancer” J Exp Clin CancerRes. 21(3 Suppl): 9-12), PI3K (Meng et al (2002) “Role of PI3K and AKTspecific isoforms in ovarian cancer cell migration, invasion andproliferation through the p70S6K1 pathway” Cellular Signaling 18(12):2262-2271), and growth factor and angiogenic signaling pathways (Maihleet al (2002) “EGF/ErbB receptor family in ovarian cancer” Cancer TreatRes. 107: 247-58; Le Page et al (2006) “Gene expression profiling ofprimary cultures of ovarian epithelial cells identifies novel molecularclassifiers of ovarian cancer” British Journal of Cancer 94: 436-445;Birrer et al (2007) “Whole genome oligonucleotide-based arraycomparative genomic hybridization analysis identified Fibroblast GrowthFactor 1 as a prognostic marker for advanced-stage serous ovarianadenocarcinomas” Journal of Clinical Oncology 25(16): 2281-2287; Trinhet al (2009) “The VEGF pathway and the AKT/mTOR/p70S6K1 signalingpathway in human epithelial ovarian cancer” British Journal of Cancer100: 971-978; and Lafky et al (2008) “Clinical implications of theErbB/epidermal growth factor (EGF) receptor family and its ligands inovarian cancer” Biochim Biophys Acta. 1785(2): 232-65).

We previously identified opioid binding protein cell adhesion molecule(OPCML) as epigenetically inactivated in 83% of ovarian cancers anddemonstrated that it was a functional tumor suppressor in vitro and invivo (Sellar et al (2003) “OPCML at 11q25 is epigenetically inactivatedand has tumor-suppressor function in epithelial ovarian cancer” Nat.Genet. 34(3): 337-43). Here, we show that OPCML interacts with anddownregulates HER2 and FGFR1 proteins, leading to inhibition of thosesignaling pathways, with consequent inhibition of in-vitro growth inSK-OV-3 ovarian cancer cells. siRNA knockdown of physiologicallyexpressed OPCML in OSE-C2 normal ovarian surface epithelial cellsstrongly upregulated HER2 and FGFR1. OPCML sensitized HER2 positiveovarian cancer cells to lapatinib and trastuzumab in vitro and was agood prognostic indicator in patients with HER2 positive ovarian cancer.The finding that OPCML actively mediates negative regulation of multipleRTK pathways opens novel research avenues in normal cell and cancerbiology.

Experimental Procedures

Antibodies

The polyclonal goat and monoclonal mouse anti-OPCML antibodies werepurchased from R&D. Anti-HER2 antibodies were purchased from Calbiochem(anti-ErbB2 (Ab-4) and (3B5) mouse MAbs). Anti-EGFR antibody was fromR&D Systems. Anti-EGFR goat pAb—cat no AF-231. Phospho-specific EGFR andHER2 antibodies were purchased from AbCam. Anti-HA antibody was fromSanta Cruz Biotechnology (Santa Cruz Calif.) HRP-conjugated secondaryantibodies were from Dako. Alexa-Fluor 488 goat anti-rabbit IgG,Alexa-Fluor 555 goat anti-mouse were from Molecular Probes (Eugene,Oreg.).

Cell Culture

The SK-OV-3 derived OPCML expressing lines (SKOBS-3.5, BKS2.1 and emptyvector SKOBS-V1.2) were described previously (Sellar et al, 2003).Stimulation time courses were undertaken with 50 ng/ml human recombinantepidermal growth factor (hrEGF—Promega) following serum-starvationovernight.

Plasmid Constructs

The OPCML cDNA expression plasmids in pcDNA3.1zeo previously described(Sellar et al, 2003) were used for transient transfections. The cDNAsencoding all three Ig domains and domains 1 and 2 were generated by PCRand introduced into the bacterial GST-fusion expression vector pGEX-6P-1(GE-Healthcare) and sequenced to confirm their fidelity. VectorpIRES-AcGFP1 (Clontech) was employed in transient transfections of OPCMLcomplete cDNA. The HA-tagged Ubiquitin pRK5-HA-Ubiquitin-WT was obtainedfrom Dr. Luke Gaughan, Newcastle University, and the EGFR and HER2 cDNAin pcDNA-3.1zeo was provided by Prof. Bill Gullick, University of Kent.FGFR1 cDNA clones was provided by Prof. Graeme Guy, FGFR1 extracellulardomain clones provided by Prof. Kyung Hyun Kim.

Expression of Recombinant OPCML and FGFR Ectodomain

Recombinant proteins were produced in the BL21 bacterial cell line(Promega) as described.

Solubilisation and Refolding of Inclusion Bodies

Inclusion bodies were solubilised in denaturation buffer (8M Urea, 20 mMTris-HCl, pH 8.0, 150 mM NaCl and 10 mM DTT) to a final concentration of5 mg/ml. The suspension was centrifuged and filtered through 0.45 μmmembrane filter. Refolding of proteins was undertaken by extensivedialysis against cold PBS in 10 kDa MWCO dialysis tubing. The suspensionwas then centrifuged and filtered to remove insoluble proteinprecipitates and soluble aggregates. Protein concentrations weremonitored throughout the experiment with protein assay reagent (Bio-RadLaboratories, California) using bovine serum albumen as a standard

RNA Extraction and cDNA Synthesis

Total RNA was extracted from cell pellets using TriReagent®(Sigma-Aldrich, Dorset, UK) following their protocol. Synthesis of cDNAwas from 1 μg of RNA template with OligodT₁₅ primers (Promega, UK), byMoloney-Murine Leukaemia Virus Reverse Transcriptase (MMLV-RT) (Promega,UK) and cDNA was stored at −20° C.

qRT-PCR

Primers were designed using PerlPrimer v1.1.14 open source software.Custom oligonucleotide synthesis was carried out by Invitrogen, UK.Quantitative reverse-transcription PCR (qRT-PCR) analysis of geneexpression was carried out on an Applied Biosystems 7900HT thermalcycler using SYBR green I technology. Premixed qPCR reagent, Platinum®Quantitative PCR SuperMix-UDG with ROX (Invitrogen, UK), was used foramplification. The expression of specific genes was normalized to theexpression of the endogenous control gene HPRT1.

Co-Immunoprecipitation and Pull-Down Assays

Cell layers were washed in PBS and incubated for 30 minutes in lysisbuffer (1% TritonX-100, 10 mM Tris pH8.0, 150 mM NaCl, 2.5 mM MgCl₂, 5mM EGTA, 1 mM Na₃VO₄, 50 mM NaF and protein inhibitor cocktail (Roche).Cell Lysates were then cleared by centrifugation at 13,000 rpm for 20minutes at 4° C. and aliquots containing equal amounts of protein wereincubated with the appropriate antibody before addition of secondaryantibody conjugated to sepharose resin. Beads were then washed 3× withlysis buffer and eluted by heating for 5 minutes in 500 of SDS samplebuffer.

Pull-down assays were performed using recombinant GST-OPCML fusionproteins bound to magnetic glutathione beads (Promega). Cell lysatesprepared as for immunoprecipitation, proteins produced using TNT invitro Rabbit reticulocyte lysate expression system (Promega) orexpressed in bacteria were used analysed for interactions.

Immunofluorescent Microscopy

Cells grown on glass slides were fixed in 4% paraformaldehyde for 10minutes at room temperature. Cells were then permeabilized for 20minutes with PBS containing 0.2% Saponin prior to blocking in PBScontaining 10% goat serum, 2% albumen 2% fetal calf serum for 1 h.Slides were incubated with appropriate combinations of mAb OPCML, mAbHER2 and pAb EGFR primary antibodies for 1 h at room temperature,followed by incubation for 1 h with animal anti-mouse Alexa-555 (OPCML),animal anti-rabbit Alexa 488 (HER2) before mounting and imaging on aZeiss LSM 510 confocal microscope.

siRNA Knockdown

Endogenous OPCML was knocked down in OSE-C2 cells by transienttransfection of a specific pool of 3 siRNAs (Stealthknockdown-Invitrogen) using lipofectamine RNAiMAX reagent.

MTT Proliferation Assay

Cell proliferation assays were carried out in quadruplicate using thethiazolyl blue tetrazolium bromide (MTT) assay. Cells were plated out in96-well plates at a density of 2,000 cells/well and cultured in lowserum medium (0.25% FCS) or low serum medium supplemented with 50 ng/mlEGF. At appropriate time points, the medium was removed from cells andreplaced with 100 μl PBS and 11 μl of 5 mg/ml MTT (w/v). Cells wereincubated in this solution for 2 hours at 37° C. and the purple fomazanproduct was solubilised in 100 μl DMSO, resuspended and read on platereader at 540 nm.

Statistical Analyses

Data are expressed as mean±SEM. Differences were analysed by Fishersexact or Student's t test. P<% 0.05 was considered significant.Progression-free survival curves were estimated using the Kaplan-Meiermethod and analysed by the log-rank test. Correlation between the mRNAexpression indices of genes was analysed using Pearson's correlationanalysis.

Statistical Analysis and Mining of Tothill Data

Gene expression data on the 251 epithelial ovarian cancers within 285ovarian tumors (published by Tothill et al (2008) Clinical CancerResearch 14: 5198) were obtained from the Gene Expression Omnibus (GEO).OPCML, EGFR and ERBB2 gene expression Pearson correlation coefficientswere computed for all probe-sets. For survival analyses included allpatients followed up to 5-years, and excluded patients withborderline/low malignant potential histology in view of their distinctnatural history compared to invasive tumors. The effect of geneexpression (probe: OPCML 206215_at, ERBB2 210930_s_at) on survival wasassessed as a continuous variable using Cox-regression, and aftertransformation to categorical variables by median dichotomization orquartiles using Kaplan-Meier curves and the log-rank test.

Results

OPCML is Rapidly Induced by EGF and FGF 1/2

Serum starved SK-OV-3 cells (low OPCML expression) {Sellar, 2003 #2}were stimulated with 50 ng/ml EGF or 10 ng/ml FGF. EGF induced OPCMLrapidly, achieving maximal mRNA expression at 30 min, with return tobasal levels of expression by 60 min (FIG. 1A(i)), with maximal OPCMLprotein at 60 min (FIG. 1A(ii)). Similarly, FGF1/2 also induced OPCMLmRNA, by 15 minutes (FIG. 1B(i)) with protein peaking at 90 minutes(FIG. 1B(ii)). These data were replicated for several other cell lines(data not shown). Specifically, induction of OPCML expression in a panelof ovarian cancer cell lines upon EGF stimulation (50 ng/ml)demonstrated consistent induction of OPCML mRNA by 5 to 10-fold, withvaried timescale of peak induction.

OPCML Interacts with HER2 and FGFR1 Via Different Binding Sites

To determine if OPCML interacted with RTKs, co-immunoprecipitation(co-IP) using an OPCML polyclonal antibody was performed in a SK-OV-3cell lines stably transfected with OPCML (BKS2.1) and vector-onlycontrols (SKOBS-V1.2). Other OPCML stable transfected clones have beenreported previously and behave identically as BKS2.1 (Sellar et al,2003). Immunoblotting with anti-HER2 and anti-EGFR demonstrated thatboth interacted with OPCML, however reciprocal Co-IP using anti-HER2 andanti-EGFR antibodies confirmed the Co-IP only for OPCML with HER2 andnot with EGFR (FIG. 2A(i & ii)). We further used GST/OPCML domain fusionproteins in pull-down experiments with either SK-OV-3 cell lysates(expressing HER2 and EGFR) or with purified TnT HER2ECD fragments(structures shown in FIG. 2B). HER2 interacted with a full length OPCMLextracellular domain (ECD) fused to GST (GST-OPCML D1±2+3) but not thetruncated OPCML ECD lacking Ig domain 3 (GST-OPCML D1±2) from SK-OV-3lysates (FIG. 2C(i)), in addition to in vitro translated HER2ECD (FIG.2C(ii)), suggesting that the third (juxtamembrane) Ig domain (Ig-III) ofOPCML is crucial for interaction with HER2. We then explored whetherOPCML interacted with the fibroblast growth factor receptors 1 and 2(FGFR1 & 2). Co-IP of SKOBS-V1.2 and BKS2.1 with OPCML antibody revealedthat OPCML bound to FGFR1, confirmed by reciprocal co-IP (FIG. 2A(ii)).We used the GST/OPCML fusion proteins in pull-downs using cell lysatestransiently transfected with full length FGFR1 (FIG. 2C(i)) andseparately in in vitro studies with His-tagged FGFR1 (FIG. 2C(ii)).These experiments showed that both GST-OPCML D1+2+3 and GST-OPCML D1+2interacted with FGFR1, therefore domain 3 was not essential for FGFR1binding, implying that FGFR1 and HER2 bound to different sites on OPCML.Further experiments showed that GST-OPCML D2+3 interacted with FGFR1 butnot GST-OPCML D3, showing that domain 2 is essential for FGFR1 binding(data not shown).

OPCML Downregulates HER2 and FGFR1, and Abrogates Phosphorylation of HERand EGFR, Together with Downstream Signaling of the MEK-ERK Cascade

We then explored the functional consequences of these OPCML-RTKinteractions. OPCML expressing BKS2.1 demonstrated strong downregulationof HER2 but not EGFR protein as compared with SKOBS-V1.2 (FIG. 3A(i)),implying that OPCML specifically regulates HER2 protein. We extended ourinvestigations to the FGF receptor family and demonstrateddownregulation of FGFR1 but not FGFR2 in BKS2.1 (FIG. 3A(ii)).Immunofluorescence microscopy (IFM) confirmed that OPCML expression inBKS2.1 dramatically reduced the levels of HER2 and FGFR1 but not EGFR orFGFR2 (FIGS. 3B(i & ii) and 3E).

We explored the impact of OPCML on cellular RTK phospho-activation andsignaling in ovarian cancer cells. Phosphorylation of 2 analogousautophosphorylation sites, HER2-Y1248 and EGFR-Y1173 was abrogated inBKS2.1 (FIG. 3C(i)) (an independent OPCML stable transfectant,previously described (Sellar et al (2003)) (data not shown), and OSE-C2expressing physiological levels of OPCML (data not shown). Similarly,FGF mediated phosphorylation of FGFR1-Y766 (known to transactivatephospho lipase Cγ) was abolished in BKS2.1 cell lines (FIG. 3C(i)). Inboth EGFR and FGFR signaling systems, we noted inhibition ofphospho-PLC. and phospho-ERK 1& 2 (T202 & Y204—FIG. 3C(ii)) but notphospho-Akt S473 or T308 (FIG. 3C(iii)) suggesting that OPCMLprincipally affected the MEK-ERK cascade. These signaling findings werephenotypically confirmed in growth assays; BKS2.1 and SKOBS-3.5 linesand OSE-C2 were profoundly growth-inhibited compared with vector controlSKOBS-V1.2 (p<0.0001, student's t-test) (FIG. 3C(iv)).

To explore the physiological role of OPCML, normal epithelial cell lineOSE-C2 (OPCML expressing) was transfected with OPCML siRNA, whichabolished OPCML protein. This resulted in a strong induction of HER2 andFGFR1 (but not EGFR or FGFR2) and phospho activation of HER2-Y1248 andEGFR-Y1173 levels (FIG. 3D(ii)). Since OPCML expression andphysiological function seems to be regulated by growth factor signalingand it is downregulating at least two members of two different familiesof RTKs, we decided to extend our analysis to other RTKs. SiRNA againstOPCML was used to verify whether RTKs appearing downregulated in theOPCML-expressing lines would show reciprocal upregulation if OPCML isknocked down in OSE-C2 cells. From this analysis, in addition to HER2,HER4 also appears downregulated in both SKOBS-3.5 and BKS2.1 cells,whereas FGFR1 and FGFR3 appear downregulated in predominantly the BKS2.1line expressing five times more OPCML than SKOBS-3.5 (FIG. 3D(i)). Thereciprocal analysis of looking at RTK expression after OPCML knockdownrevealed HER2, HER4, FGFR1, all showing substantive upregulation insiRNA lane. FGFR3 exhibits a slight increase in expression level withknockdown (FIG. 3D(ii)). In contrast OPCML does not affect EGFR, HER3,FGFR2, FGFR4, EPHA50, VEGFR1 and VEGFR3.

OPCML Prevents HER2/EGFR Hetero Dimer Formation and Reduces EGF ReceptorAvailability.

SKOBS-V1.2 and BKS2.1 cell extracts were subjected to Co-IP andimmunoblotted with antibodies as shown in FIG. 4A, demonstrating loss ofhetero-dimerisation in the presence of OPCML. Further, OPCML reduced EGFreceptor availability (FIG. 4B).

OPCML is Localized in the Detergent-Resistant (Raft) Membrane Fractionand Co-Localizes with EGFR and HER2 in Ovarian Cancer Cells.

To define the mechanism of OPCML-based RTK degradation, we used HER2 asa paradigm for further study. Initially, we investigated the influenceof OPCML expression upon the mode of HER2 degradation linked toimmunofluorescent confocal microscopy (IFM) analysis to examine thetrafficking of OPCML and HER2 in cells. It has been previously reportedthat GPI-anchored proteins are sequestered in the detergent insoluble‘lipid-raft’ membrane microdomain of cells (Sangiorgio et al (2004) ItalJ Biochem 53(2): 98-111). To examine the localisation of OPCML (a GPIanchored protein) within lipid rafts, purified membrane of OPCMLnegative (SKOBS-V1.2) and positive (BKS-2.1) were subjected tosolubilisation in 1% Triton X100 (for detailed method see Materials andMethods) and samples subjected to ultracentrifugation to separatedetergent solubilised and insoluble proteins (FIG. 5A(i)). Thisexperiment revealed that the majority of OPCML was localized within thedetergent insoluble fraction, along with Caveolin-1 (a marker ofcaveolae—a distinct form of lipid raft domain). Interestingly, HER2, inthe OPCML-expressing line, was reduced as previously shown in FIGS. 3Aand 3B but also sequestered in the detergent insoluble fraction whencompared to the OPCML negative line, where HER2 was equally distributed.The distribution of EGFR was only marginally effected by the expressionof OPCML. IFM was employed to examine the trafficking of OPCML in cells;EEA-1 (a marker of the early endosome) and caveolin-1 (a marker of theraft-caveolar pathway) were used to distinguish between Clathrin-coatedpit and caveolar endocytic vesicles. These studies revealed that themajority of the internalized protein co-localized with Caveolin-1compared to EEA-1 (OPCML+ cell line: cav-1 co-localisation=23%,EEA-1=7.5% of total HER2; OPCML− cell line: cav-1 co-localisation=4.5%,EEA-1=32% of total HER2). Furthermore, vesicular staining was seen to bemarkedly different within the cell, consistent with these representingdistinct compartments (FIG. 5A(ii)). IFM also confirmed that OPCMLco-localized with EGFR and HER2 in ovarian cancer cells (FIG. 5A(iii)).

We next transfected both OPCML-expressing and non-expressing cell lineswith a HA-tagged ubiquitin construct to analyze the levels of receptorubiquitination +/−OPCML. Twenty four hours post transfection, cells wereserum starved and subjected to acute stimulation with EGF (50 ng/ml) for60 minutes. Consistent with the significant reduction in receptorlevels, OPCML expression was associated with enhanced ubiquitination ofHER2, which was strongly increased upon EGF stimulation (FIG. 4B(i&ii)).IFM and quantification of co-localisation demonstrated that OPCMLexpression induced a shift in proportion of the HER2 into caveolin-1positive vesicles compared to a predominant co-localisation with EEA-1in the OPCML negative cell line (+OPCML: HER2/CAV-1, 22.863%±1.859;HER2/EEA-1 8.767±1.852. −OPCML: HER2/CAV-1, 4.767%±1.559; HER2/EEA-130.667±3.756) (FIG. 5C(i&ii)). Transmembrane proteins in the EEA-1compartment can enter either the late-endosome-lysosome for degradation,or the Rab11-positive recycling endosome. Whilst Caveolin-1 positivevesicles have been reported to be non-recycling and result inproteasomal degradation of their cargo (Di Guglielmo et al (2003) NatCell Biol 5: 410-421). Consistent with degradation by the proteasome,chloroquine (CQ), a weak base that alkalinises the lysosome, wasineffective, but MG-132, a potent antagonist of the proteasomal 26Sproteinase, inhibited HER2 degradation in the OPCML expressing cell linewith no effect on EGFR expression found (FIG. 5D(i&ii)). Furthermore,disruption of cholesterol using methyl-β-cyclodextrin (Mβ-CD) alsoinhibited the degradation of HER2 and increased the phosphorylation atY1248 (FIG. 5D(iii)) suggesting an important role for the lipid-raft inthe OPCML-specific regulation and degradation of HER2. In conclusion,OPCML binds specifically to HER2, sequesters the receptor inlipid-rafts, enhancing caveolar-based endocytosis, ubiquitination andsubsequent proteasomal degradation of the oncogenic receptor.

OPCML Regulates/Predicts Response to Lapatinib in Ovarian and BreastCancer.

The finding that OPCML could regulate activity of HER2 and EGFR led usto explore whether OPCML might influence the efficacy of anti-EGFR/HER2therapeutics. OPCML transfected and control cells were pre-incubatedwith lapatinib, trastuzumab, cituximab, erlotinib and gefitinib. We thenused EGF induced phospho-ERK activation as an assay to define theeffectiveness of therapeutic inhibition. The dual inhibitor of EGFR andHER2 tyrosine kinases, lapatinib, exhibited strong OPCML mediatedsensitization, reducing the effective concentration of lapatinibrequired to abolish the phospho-ERK signal by 10-fold for BKS2.1compared with SKOBS-V1.2 (FIGS. 6A(i) and 6C). We noted enhanceddown-regulation of phospho-AKT in OPCML expressing cells. Lessersensitization than for lapatinib was observed with trastuzumab (FIG.6A(ii)). Notably, cetuximab, erlotinib and gefitinib, inhibitors of EGFRshowed no sensitization in OPCML transfected cells (data not shown)consistent with the hypothesis that OPCML interacts with HER2 and notEGFR.

We then investigated whether siRNA knockdown of physiological OPCMLexpression in normal OSE-C2 cells could affect sensitivity to lapatinib.We observed that the lapatinib-mediated reduction in phospho-ERK signalstrength was significantly reversed by OPCML siRNA knockdown in thesenormal ovarian surface epithelial cells (FIG. 6B). These datademonstrate that OPCML modulates sensitivity to lapatinib throughregulation of the level of HER2, however the mechanism of this findingremains to be clarified.

We next tested whether OPCML could be used to predict response tolapatinib in ovarian and breast cancer. Histology was obtained by newbiopsy of recurrent disease and TTP (time in months to progression fromstart of therapy until progression) assessed. Docetaxel andanthracyclines were administered for a maximum of 6 cycles andcapecitabine was administered until disease progression or unacceptabletoxicity. HER2 immunohistochemistry (IHC) was performed using the DakoHerceptest kit: 3+ in all cases. The results are shown in Table 1 below.

TABLE 1 Case Response num- Previous treatment chronology to OPCML berHistology (TTP in months)* lapatinib Score 1. G2 IDC, ER Adjuvant:antracyclines and SD − 6/8, PR 0/8, taxanes (23) then adjuvant HER2 3+trastuzumab (12) Metastatic: Hormonal therapy (12) Capecitabine (1) 2.G3 IDC, Adjuvant: anthracyclines (48) SD − ER/PR (0/8), Metastatic:Trastuzumab (8) HER2 3+, Capecitabine (2) Vinorelbine (8) Taxanes (5) 3.G3 IDC, Metastatic: Anthracyclines (12) PD + ER++, PR++, Hormonaltherapy (12) HER2 3+ Trastuzumab (5) (metastatic Taxanes (5)presentation) Capecitabine (1) 4. G3 IDC, ER Metastatic: Capecitabine(14) PD + (8/8), PR Trastuzumab (5) (8/8), HER2 Vinorelbine (4) 3+(metastatic Taxanes (4) presentation) Hormonal therapy (4) 5. G3 ILC,Metastatic: Anthracyclines (3) PR ++ ER/PR (0/8), Taxanes (4) HER2 3+Trastuzumab (4) (metastatic Gemcitabine and presentation) vinorelbine(7) Capecitabine (1) 6. G2 IDC, Adjuvant: anthracyclines (7) PR ++ ER/PR(0/8), Metastatic: Taxanes (10) HER2 3+ Trastuzumab (12) Vinorelbine (9)7. G2 IDC, Adjuvant: anthracyclines (36) PR ++ ER/PR (0/8), Metastatic:Trastuzumab (5) HER2 3+ Taxanes (4) Capecitabine (2) Vinorelbine (4) 8.G3 IDC, Adjuvant: Anthracyclines and PR +++ ER/PR (0/8), taxanes (14),trastuzumab (8) HER2 3+, inflammatory Estrogen receptor (ER) andprogesterone receptor (PR) are either scored using H scores (out of 8)or IHC. IDC = invasive ductal carcinoma, ILC = invasive lobularcarcinoma. SD = stable disease, PR = partial response, PD = progressivedisease, by RECIST criteria. Examples of OPCML IHC − to +++ are shown.OPCML is a Prognostic Factor in Strongly HER2 Expressing Ovarian Cancer.

In view of the strong tumor suppressor role of OPCML and these findings,we explored whether its expression was related to ovarian cancerprognosis. We used a recently published expression microarray dataset of251 ovarian cancers (Tothill et al, 2008) with full clinical annotationand follow-up of patients for progression free survival (PFS). Therelationship between OPCML mRNA expression and PFS was examined for all251 ovarian cancer patients with epithelial ovarian cancers in thedataset. Overall high OPCML expression demonstrated a significantassociation with better survival, as shown by the Kaplan-Meier curve inFIG. 7A(i) (Log-rank p=0.061 Breslow test p=0.034), although thedifference was of modest magnitude. However, because the findingsdescribed herein suggested that OPCML's tumor suppressor role wasrelated to repression of HER2 and FGFR1 function we specificallyanalysed the patient cohort according to their HER2RNA expression andexplored the impact of OPCML expression on this group of patients. Wefound that OPCML mRNA level was strongly prognostic only for patientsexpressing top quartile HER2 mRNA levels; patients with above medianexpression of OPCML had 27 months median PFS (FIG. 7A(ii)) and Table 1 &2) compared with top quartile HER2 and below median OPCML expressionwith 13 month median PFS (Log-Rank p=0.004). In contrast, bottomquartile HER2 expressing patients showed similar survival regardless ofOPCML expression. This validated our hypothesis that OPCML impacts on anintact HER2 pathway. No significant association was observed for EGFR orFGFR1 and OPCML with PFS in this dataset (data not shown).

A possible explanation for this clinical data is that strong OPCMLexpression (in the context of strong HER2 expression) regulates HER2protein level/activity and abrogates HER2 pro-oncogenic signaling withconsequent better patient prognosis, whereas tumors with weak OPCMLexpression and strong HER2 expression have unrestrained HER2pro-oncogenic signaling and consequently poor prognosis.

Discussion

OPCML therefore has repressor-adaptor function, interacting with HER2and FGFR1, targeting them for degradation and thereby physiologicallynegatively regulating both the EGF and FGF signaling pathways in normalovarian surface epithelium, and conversely, by CpG island somaticmethylation, activating both these pathways concurrently in epithelialovarian cancer.

In summary the OPCML tumor suppressor functions by concurrentlynegatively regulating HER2 and FGFR1 in normal ovarian surfaceepithelial cells and in ovarian cancer. This finding has generalimplications for understanding the relationship of IgLONs to the RTKpathways, and their role in both biology of ovarian surface epithelialcells and also in the understanding of how somatic methylation of thistumor suppressor uncovers such strong pro-oncogenic phenotype driven byseveral RTK pathways.

Table 2a and b: show case processing summary, means and median survivaland data comparisons for the data depicted in Kaplan Meier curves (FIG.7(i) and (ii)).

Table 2A Case Processing summary B OPCML Censored 215 Total No No ofEvents No Percent Low OPCML 122 93 29 23.80% High OPCML 129 91 38 29.50%Overall 251 184 67 26.70% Means and Medians for Survival Time Mean(a)Median 95% confidence interval 95% confidence interval B OPCML 215Estimate Std. Error Lower Bound Upper Bound Estimate Std. Error LowerBound Upper Bound Low OPCML 20.788 1.652 17.55 24.027 14 0.625 12.77515.225 High OPCML 24.771 1.669 21.5 28.043 19 1.101 16.843 21.157Overall 22.866 1.187 20.539 25.193 15 1.138 12.77 17.23 (a)Estimation islimited to the largest survival time if it is censored OverallComparisons Chi- Square df Sig. Log Rank (Mantel-Cox) 3.522 1 0.061Breslow (Generalised Wilcoxon) 4.502 1 0.034 Tarone-Ware 4.451 1 0.035Test of Equality of survival distribution for the different levels ofB_OPCML_215 Table 2B Case Processing summary Censored Q-HER2-930 TotalNo. No. of Events No. percent Low HER2/Low OPCML 41 38 3 7.30% LowHER2/High OPCML 21 17 4 19.00% High HER2/Low OPCML 17 13 4 23.50% HighHER2/High OPCML 42 26 16 38.10% Overall 121 94 27 22.30% Means andMedians for Survival Time Mean(a) Median 95% confidence 95% confidenceinterval interval Q-HER2-930- Lower Upper Std. Lower UpperOPCML-215-10-11-40-41 Estimate Std. Error Bound Bound Estimate ErrorBound Bound Low HER2/Low OPCML 16.016 1.73 12.624 19.407 13 1.232 10.58615.414 Low HER2/High OPCML 17.026 2.393 12.337 21.716 12 2.511 7.07916.921 High HER2/Low OPCML 16.243 2.909 10.541 21.946 13 1.159 10.72815.272 High HER2/High OPCML 30.097 3.299 23.63 27 27 5.753 15.725 38.275Overall 21.479 1.614 18.315 15 15 1.47 12.119 17.881 (a)Estimation islimited to the largest survival time if it is censored OverallComparisons Chi-Square df Sig. Log Rank (Mantel-Cox) 13.535 3 .004Breslow (Generalized 9.200 3 .027 Wilcoxon) Test of equality of survivaldistributions for the different levels ofQ_erb_930_Opcml_215_10_11_40_41.Example 2: Exogenous OPCML Inhibits Receptor Tyrosine Kinase Signalingand Ovarian and Breast Cancer Cell Growth In Vitro, While Sparing NormalOvarian Surface Epithelial Cells

To complement the findings described in Example 1, we expressed andpurified recombinant human OPCML and assessed its affect on in vitrotyrosine kinase signaling and cell growth. The results are in agreementwith those in Example 1.

FIG. 8 shows purification of recombinant human OPCML expressed in E.coli. Expressed OPCML was present in inclusion bodies and wassuccessfully refolded by dialysis into PBS. Recombinant OPCML is a 272amino acid polypeptide whereas physiologically synthesized OPCML is a345 amino acid polypeptide. Post-translationally modified OPCML(including N-linked glycosylation) is 55 kDa, whereas withoutglycosylation, the signal peptide region and the GPI anchor region,OPCML is 31 kDa. The protein and polynucleotide sequences of recombinanthuman OPCML correspond to SEQ ID Nos: 5 and 6 respectively.

FIG. 9 shows cellular uptake of recombinant OPCML. Cells which took upexogenous OPCML demonstrate downregulation of HER2, as confirmed by IFM.

FIG. 10 shows that administration of exogenous OPCML inhibits receptortyrosine kinase signaling in vitro. Specifically, administration ofexogenous OPCML downregulates HER2 and ERK protein levels.

FIGS. 11A and 11B show that administration of exogenous OPCML inhibitsSKOV-3 cell growth in vitro, as assessed by a MTT cell growth assay.

FIG. 11C shows that OPCML inhibits growth of a range of ovarian andbreast cancer cell lines, while sparing normal ovarian surfaceepithelial cells. Interestingly, growth of HER2+ and HER2− breast cancercell line was profoundly inhibited, suggesting that the mechanism is notsolely restricted to HER2+ cells but may be mediated through FGFRpathways or other EGFR components that interact with HER2 and arephospho-inactivated as part of OPCML therapy. Only one cell line showedresistance to OPCML (PEA2).

Example 3: OPCML is a Prognostic Factor in Breast, Lung and GliomaCancers

Having established that OPCML is a prognostic factor in strongly HER2expressing ovarian cancer (see Example 1), we assessed whether itsexpression was related to the prognosis of other cancers. This was doneby a Kaplan Meier analysis of overall survival according to OPCMLdichotomized survival.

FIG. 12A (first graph—A1) shows that OPCML expression is a goodprognostic factor in EGFR/HER2 positive node negative breast cancers inpatients receiving no adjuvant systemic therapy. OPCML expressing tumorsshow better survival, with a 10 year relapse free survival of 72% vs52%. (‘cum survival’=cumulative survival). Analysis was based on apublished dataset of 149 node negative breast cancer patients (Wang eta/2005, Lancet 365(9460): 671). The second two graphs (A2 and A3) showthat OPCML expression is a particularly good prognostic factor in ER—breast cancer.

FIG. 12B shows that OPCML is a prognostic factor in lung cancer. OPCMLexpressing tumors show better overall survival (OS). Analysis was basedon a published dataset of 115 lung cancer patients (Takeuchi et al 2006,J Clin Oncol 24(11): 1679-1688).

FIGS. 12C and 12D show that OPCML is a prognostic factor in brain highgrade gliomas. OPCML expressing tumors show better overall survival(OS). Analysis was based on a published dataset of high grade gliomapatients (Phillips et al2006 Cancer Cell 9(3): 157-73).

Example 4: Functional Effects of OPCML Expression in SKOV3 In Vitro andIn Vivo

We next investigated the effects of OPCML expression in SKOV3 cells invivo, using methods described in Sellar et al (2003).

FIG. 13A shows the effect of OPCML expression on in vitro growth ofSKOV3 cells, with OPCML expression inhibiting growth relative to normalSKOV3 cells or to SKOV3 cells in which OPCML expression wasknocked-down, in agreement with the data described above.

FIGS. 13B and 13C show the effect of OPCML expression on in vivo growthof SKOV3 cells. OPCML expression reduced mean tumor volume relative tonormal SKOV3 cells or to SKOV3 cells in which OPCML expression wasknocked-down.

Example 5: Further Studies on the Role of OPCML

Summary

OPCML, a GPI anchored tumor suppressor gene is inactivated by somaticmethylation in multiple cancers. We previously identified this gene byLOH mapping and demonstrated that it was inactivated by somaticmethylation in 80% of ovarian cancers. Restoring OPCML expression bystable transfection suppressed in-vitro growth and in-vivotumorigenicity. We investigated the role of OPCML in growth signalingpathways. In SKOV-3 and PEO1, ovarian cancer cell lines with noexpression of OPCML, we demonstrated that OPCML negatively regulates aspecific repertoire of receptor tyrosine kinases (RTKs) EPHA2, FGFR1,FGFR3, HER2 and HER4, and reciprocally, OPCML siRNA knockdown in normalovarian surface epithelial cells up-regulates these same RTKs. OPCML hasno effect on the RTKs EPHA10, FGFR2, FGFR4, EGFR, HER3, VEGFR1 andVEGFR3. Example immunoprecipitation experiments revealed that OPCMLbinds to EphA2, FGFR1 and HER2 extracellular domains with no suchinteraction to EGFR, thus OPCML binds directly to RTKS that itnegatively regulates. We demonstrate that OPCML is located exclusivelyin the raft membrane fraction and sequesters RTKs that it binds to theraft fraction, leading to polyubiquitination and proteosomal degradationvia a cav-1 endosomal mechanism resulting in systems depletion of thisspecific RTK repertoire, that does not occur with RTKs that OPCML doesnot bind. We demonstrate that OPCML abrogates EGF mediatedphosphorylation of FGFR1, HER2 and EGFR and the downstreamphosphosignaling of pErk and pAKT.

A recombinant modified OPCML-like protein without a GPI anchor, signalpeptide or glycosylation was constructed and expressed in E. coli. ThisrOPCML tumor suppressor protein therapeutic caused growth inhibition byapoptosis in 6/7 ovarian cancer cell lines tested, with no effect onOPCML expressing normal ovarian surface epithelium, by an identicalmechanism to the transfected normal protein. rOPCML was then injectedintraperitoneally twice weekly in two murine intraperitoneal models ofovarian cancer (nude mouse A2780 and SKOV3) and demonstrated profoundinhibition of tumour weight, ascites volume and peritoneal disseminationcompared with BSA control.

Mechanism of OPCML TSG Function

OPCML is a non-transmembrane, external lipid leaflet GPI-anchoredprotein, and is frequently lost from cells by somatic inactivation ofthe gene. We hypothesised that it may mediate its tumour suppressorproperties via interactions with transmembrane signalling proteins, andso we analysed the effect of receptor tyrosine kinase (RTK) growthfactor stimulation on OPCML gene expression. Treatment of 4/4 ovariancancer cell lines with EGF or FGF 1/2 resulted in rapid OPCML RNA andconcomitant protein expression (data not shown) suggesting that OPCMLmaybe a putative suppressor-type immediate-early negative feedbackregulator.

Stable transfection of OPCML in the basal unstimulated orligand-stimulated SKOV-3 ovarian cancer cells, resulted in the profoundprotein down-regulation of a specific repertoire of RTKs: EPHA2; FGFR1;FGFR3; HER2 and HER4 (FIG. 16A) and this RTK down-regulation spectrum isreproducible by transient transfection of a different ovarian cancercell line, PEO1 (FIG. 16B). These same RTKs were also reciprocallyup-regulated when physiological OPCML was knocked down by siRNA inOSE-C2, a normal ovarian surface epithelial cell line (Davies et al,(2003) Experimental Cell Research 288: 390-402) (FIG. 16C). Thisspecific inactivation by OPCML was not seen for other RTKs we haveinvestigated so far including: EPHA10; FGFR2; FGFR4; EGFR; HER3; VEGFR1and VEGFR3 (FIG. 16). The phenotypic consequences of these signallingeffects were confirmed in growth assays in ligand-supplemented mediawhere OPCML-transfectants were significantly growth-inhibited comparedwith vector control (data not shown).

Negative Regulation of Specific RTKs by OPCML is Related to DirectProtein Interaction

We further explored as examples EPHA2, FGFR1 and HER2, RTKs that arestrongly inactivated at the protein level upon OPCML expression. We alsoanalysed EGFR as an example of a protein that is unaffected by OPCML.Immunoprecipitation (IP) experiments demonstrated protein/proteininteractions with EPHA2, FGFR1 and HER2, but no such binding to EGFR(FIG. 17A). These findings were further confirmed using a recombinantOPCML (GST-OPCML D1-3) pull-down assay (FIG. 17B) which was then used todetermine that the extracellular domains (ECDs) of the RTKs FGFR1 andHER2 (as examples) were capable of interacting specifically with OPCML(FIGS. 17C&D), showing that the site of interaction lay within the ECDof the RTKs and domain 1-3 of OPCML, defining the site of OPCML actionas extracellular.

Downstream Signalling

Upon acute ligand stimulation, OPCML expression led to profoundabrogation of phospho-FGFR1-Y766, phospho-HER2-Y1248 and, also,phospho-EGFR-Y1173. Whilst EGFR total protein down-regulation is NOTobserved, presumably due to the absence of an RTK ECD physicalinteraction with OPCML, the consequence of OPCML mediated loss of theactivating dimerisation partners of EGFR, (HER2 and HER4), coupled withthe continuing availability of the HER3 family member (that results inan inhibitory dimerisation with EGFR), explain the down-regulation ofEGFR signalling even though total EGFR levels are unaffected (FIG. 18A).Analysis of FGFR1 signalling showed a similar pattern of phosphoinhibition relating to protein down-regulation (FIG. 18B).

Analysis of downstream signalling demonstrated abrogation of phospho-ERK1 & 2 (T202 & T204) and phospho-AKT-S473 (FIG. 18C), suggesting thatboth pro-growth and pro-survival pathways are inhibited by OPCMLre-expression, via a systems level abrogation of this specific RTKspectrum.

OPCML-Mediated RTK Degradation Mechanism

Using HER2 as a paradigm molecule of OPCML-RTK regulation, we found thatthe available HER2 in OPCML expressing cells was sequestered in thedetergent resistant membrane (DRM) fraction. In the OPCML non-expressingline, HER2 was found equally distributed between the DRM and thedetergent soluble (non-raft) fractions. The total level of EGFR was notaffected by the expression of OPCML and its distribution showed a muchless pronounced but discernible shift to the DRM fraction (FIG. 19A).These data indicate that OPCML expression leads to redistribution ofHER2 to the DRM fraction in the plasma membrane (that broadly correlateswith membrane “rafts”). IFM was employed to examine the trafficking ofOPCML in cells; EEA-1 (a marker of the early endosome) and Caveolin-1 (amarker of the raft-caveolar pathway) were used to investigate thisapparent redistribution. A decrease in HER2 co-localisation with EEA1shows that the sequestration of HER2 to the DRM fraction decreases itsendocytosis via clathrin-mediated pathways. While an increase inco-localisation with caveolin-1 was observed, the immunofluorescencepattern suggests this is a function of the redistribution of HER2 intothe DRM fraction (housing lipid-raft domains) where caveolin is alsolocalised, as HER2 did not appear to be exclusively localised tocaveolae in the presence of OPCML expression. Furthermore, in thepresence of OPCML the staining was organised into specific sub-cellularparticles, suggestive of distinct vesicular compartments (FIGS. 19B&C).

This analysis demonstrated that OPCML expression was associated withincreased ubiquitination of HER2 (that binds OPCML), which was stronglyincreased upon EGF stimulation (FIGS. 19D&E). Exposure to MG-132, apotent inhibitor of the proteasomal 26S proteinase, attenuated HER2degradation with no such effect on EGFR expression (that does not bindOPCML). In contrast, chloroquine, a weak base that alkalinises thelysosome, showed no inhibition of HER2 degradation (FIGS. 19H&I). Thissuggested that the proteasomal pathway was preferentially utilised forOPCML-mediated HER2 degradation. Furthermore, disruption of cholesterol(a component of DRM fraction/lipid rafts) using methyl-β-cyclodextrin(Mβ-CD) also inhibited the degradation of HER2 and increased HER2phosphorylation (FIG. 19J) suggesting that cholesterol-rich lipid-raftstructures are important for OPCML-specific internalisation anddegradation of HER2.

These findings suggest that OPCML-mediated negative regulation of thisspecific repertoire of RTKs is the result of direct binding of OPCML tothe ECD of that RTK. These multiple but specific binding events resultin ‘lipid-raft’ sequestration, enhanced ubquitination, and a switch awayfrom clathrin-mediated endocytosis to proteasomal degradation of thosespecific RTKs negatively regulating their signaling through reducingtheir protein level. Our data, in the context of very recentpublications (Howes et al (2010) J. Cell Biol. 190(4): 675-91; Howes etal (2010) Curr. Opin. Cell Biol. 22(4): 519-527)), would suggest thatCLIC/GEEC bulk internalization route is a strong candidate pathway forOPCML-mediated degradation of HER2 and that this is linked to RTKinactivation and the observable strong tumour suppressor phenotype ofOPCML.

Recombinant OPCML (r-OPCML) Inhibits Tumour Growth In Vitro and In Vivo

Purified recombinant human OPCML domain 1-3 protein (r-OPCML) (FIG. 8)was produced from the bacterial expression vector (pHis-Trx) subclonedwith domains 1-3 of OPCML, excluding the signal peptide and GPI anchorsequences (FIG. 8A). Addition of r-OPCML protein to growth mediademonstrated a specific, dose-dependent inhibition of cell growth inOPCML non-expressing SKOV-3 ovarian cancer cells, without affectingnormal ovarian surface epithelial cells, OSE-C2 (FIG. 20A). We haveconfirmed that r-OPCML profoundly inhibited cell growth in 6 of 7additional OPCML non-expressing epithelial ovarian cancer cell lines; 2of 2 breast HER2-positive and negative cells; and 5 of 5 lung cancercell lines (FIG. 20B). To determine the mechanism of thispharmacological growth inhibition we performed Annexin V FACS apoptosisassay in SKOV-3 and A2780 demonstrating evidence of early apoptosisinduced by r-OPCML at 2-6 hours post exposure depending on cell line(FIG. 20C). We then performed caspase-glo apoptosis assays across aconcentration range in SKOV-3 and A2780 ovarian cancer cells anddemonstrated that r-OPCML induces apoptosis in both these cell lines ina dose dependent fashion, demonstrating the underlying mechanism of theobserved growth inhibition (FIGS. 20D&E). Immunoblotting confirmed thataddition of r-OPCML protein to media potently downregulated the samespectrum of RTKs as seen by transfecting OPCML into cancer cells, aswell as abrogating pERK and pAKT in both SKOV-3 and A2780 (FIG. 21).This suggests that pharmacological use of extracellular unanchoredr-OPCML utilises the same mechanism of action as transfection inducedintracellular re-expression of the normal GPI-anchored, glycosylated,OPCML protein. These data were confirmed by IFM for HER2 in SKOV-3,closely mirroring stable transfection of the normal protein in the samecell line (see, FIG. 9).

In view of these in-vitro findings, we proceeded to determine whetherr-OPCML protein had potential and relevance as an in-vivo tumoursuppressor therapy. Mice with either SKOV-3 or A2780 cancer cellsinjected intraperitoneally (IP), after tumour establishment, receivedtwice-weekly IP injections of either 1 ml (10 μM) bovine serum albumin(BSA) or 1 ml (10 μM) r-OPCML. The experiment was terminated after 3weeks due to obvious extensive IP tumour growth and deterioratingcondition of BSA-treated control animals whereas r-OPCML treated miceremained well (FIG. 22A). r-OPCML significantly and profoundlysuppressed both IP tumour growth and ascites formation in-vivo in bothIP models (FIGS. 22B-D), and in A2780 tumour bearing mice, profoundlyinhibited the number of IP peritoneal deposits compared with BSA control(FIG. 22E). Western blotting of SKOV3 IP tumour recovered from BSAtreated and r-OPCML treated animals clearly demonstrated the samespectrum of RTKs inhibited as predicted from the in-vitro analysis (FIG.22F).

Conclusion

OPCML mediates its tumour suppressor function by systems level negativeregulation of at least 5 RTKs and a recombinant modified OPCMLderivative is a potent tumor suppressor protein therapeutic in-vitro andin-vivo.

The invention claimed is:
 1. A pharmaceutical composition comprising afragment of OPCML consisting of residues 39-219 of human OPCML (SEQ IDNO:1) or residues 39-310 of human OPCML (SEQ ID NO:1), and apharmaceutically acceptable carrier, diluent or excipient, wherein thefragment of OPCML lacks OPCML signal sequence residues 1-27, and whereinthe fragment is fused to a fusion partner, wherein the fusion partner isa non-OPCML polypeptide selected from the group consisting of anantibody Fc fragment and peptide tag.
 2. The pharmaceutical compositionof claim 1, wherein the fusion partner is an antibody Fc fragment. 3.The pharmaceutical composition of claim 1, wherein the fragment lacksOPCML GPI anchor residues.
 4. The pharmaceutical composition of claim 3,wherein the fusion partner is an antibody Fc fragment.